Method of selectively measuring triglycerides

ABSTRACT

The present invention relates to a reagent for selective measurement of triglycerides contained in very low density lipoprotein and intermediate density lipoprotein or in very low density lipoprotein in a test sample, including a first reagent that contains a first selective reaction promoter, which is an ether or ester compound of a polyoxyalkylene capable of reacting lipoprotein lipase selectively with triglycerides contained in low density lipoprotein and high density lipoprotein; lipoprotein lipase; enzymes which catalyze a series of reactions leading to the generation of hydrogen peroxide or a reduced coenzyme from glycerol; and an enzyme which catalyzes a reaction leading to the conversion of hydrogen peroxide or a reduced coenzyme into another substance, and a second reagent that contains a second selective reaction promoter, which is capable of reacting lipoprotein lipase selectively with triglycerides contained in very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein and high density lipoprotein and to a method for selective measurement of triglycerides contained in very low density lipoprotein and intermediate density lipoprotein or in very low density lipoprotein in a test sample which uses the above reagent.

TECHNICAL FIELD

The present invention relates to a method and reagent for selectivemeasurement of triglycerides contained in very low density lipoproteinand intermediate density lipoprotein or in very low density lipoproteinwhich is important for clinical diagnosis of arteriosclerosis.

The invention is significant in the fields of chemistry, life science,medical treatment and the like, and particularly in the field oflaboratory tests.

BACKGROUND ART

Cholesterol and triglycerides are essential nutrients for livingorganisms. They present in blood in such a form that they are packed inan amphipathic “shell” (as lipoproteins) because they are difficult todissolve in water.

There are several classes of lipoproteins: chylomicron, very low densitylipoprotein (VLDL), intermediate density lipoprotein (IDL), low densitylipoprotein (LDL) and high density lipoprotein (HDL), which constitute acomplex metabolic system.

These lipoproteins each contain cholesterol and triglycerides, and verylow density lipoprotein and intermediate density lipoprotein arecomposed primarily of triglycerides and greatly involved in thedevelopment of arteriosclerosis. It is therefore useful to separatelymeasure triglycerides in VLDL and IDL.

Large-scale follow-up surveys of factors involved in the development ofarteriosclerosis have demonstrated that LDL cholesterol and the totalamount of serum triglycerides (hereinafter, referred to as “totaltriglycerides”) promote the development of arteriosclerosis, while HDLcholesterol inhibits the development of arteriosclerosis.

Triglycerides constitute a very small part of LDL and HDL, but a largepart of chylomicron, VLDL and IDL.

And triglycerides contained in chylomicron have been found not to be arisk factor for arteriosclerosis.

There have been established and widely used several methods whichmeasure total triglycerides contained in lipoproteins without separatinglipoproteins according to class (Henry, J. B., Clinical Diagnosis andManagement by Laboratory Method, Philadelphia: W. B. Sauders, pp.189-204).

These methods involves degrading triglyceride present in serum intoglycerol by lipoprotein lipase, subsequently converting it intoglycerol-3-phosphate by glycerol kinase, and further converting it intodihydroxyacetone-3-phosphate by glycerol-3-phosphate oxidase, followedby calorimetric assay of the generated hydrogen peroxide using aperoxidase system (Trinder reaction system).

There is another method which measures total triglycerides inlipoproteins by generating NADH (a reduced coenzyme) by the action ofglycerol-3-phosphate dehydrogenase, instead of glycerol-3-phosphateoxidase, and measuring the NADH.

These methods are widely called enzymatic assays.

There have also been known methods which measure cholesterol containedin LDL or HDL by allowing a particular surfactant and additive toselectively act on LDL or HDL (e.g. JP Patent Publication (Kokai) No.9-313200, JP Patent Publication (Kokai) No. 9-285298), and such methodshave been widely used for the purpose of laboratory tests etc.

As a method for selective measurement of triglycerides in VLDL and/orIDL, however, there has been only one method, ultracentrifugation, whoseoperating procedures are complicated and there has been neither methodnor reagent for selective measurement of triglycerides in VLDL and/orIDL which is readily performable.

However, Dr. Okada, one of the inventors of this invention, has resolvedthe above described problem and accomplished a method and reagent forselective measurement of triglycerides contained in VLDL and/or IDL (WO00/60112).

DISCLOSURE OF THE INVENTION

Accordingly, the object of this invention is to establish a method andreagent which enables the selective measurement of triglyceridescontained in very low density lipoprotein (VLDL) and intermediatedensity lipoprotein (IDL) or in very low density lipoprotein (VLDL) in atest sample while ensuring ease of operation and good accuracy.

More specifically, the object of this invention is to establish a morereadily and accurately performable method and reagent for selectivemeasurement of triglycerides contained in VLDL and IDL or in VLDL in atest sample which does not require any complicated operating procedures,such as centrifugation with an ultracentrifuge, and is applicable toautomatic analyzers generally in use.

After directing tremendous research effort toward the solution of theabove described problem, the inventors of this invention have found thatselecting selective reaction promoters from among those described in WO00/60112 makes more readily and accurately performable the selectivemeasurement of triglycerides contained in VLDL and IDL or in VLDL in atest sample and finally accomplished this invention.

This invention embraces the following:

(1) A method for selective measurement of triglycerides contained invery low density lipoprotein and intermediate density lipoprotein or invery low density lipoprotein in a test sample, comprising the followingtwo steps:

a first step that comprises

1′. exposing and reacting the test sample to and with lipoprotein lipaseand some other enzymes, which catalyze a series of reactions leading tothe generation of hydrogen peroxide or a reduced coenzyme from glycerol,in the presence of a first selective reaction promoter, which is anether or ester compound of a polyoxyalkylene capable of reactinglipoprotein lipase selectively with triglycerides contained in lowdensity lipoprotein and high density lipoprotein, to generate hydrogenperoxide or a reduced coenzyme from the triglycerides contained in thelow density lipoprotein and the high density lipoprotein in the testsample,

2′. reacting the hydrogen peroxide or reduced coenzyme generated by thereaction 1′ with an enzyme which catalyzes a reaction leading to theconversion of hydrogen peroxide or a reduced coenzyme into anothersubstance, and

3′. eliminating the triglycerides contained in the low densitylipoprotein. and the high density lipoprotein by the reactions 1′ and2′, and

a second step that comprises

1′. subsequently, after the first step, reacting the test sample withlipoprotein lipase and some other enzymes, which catalyze a series ofreactions leading to the generation of hydrogen peroxide or a reducedcoenzyme from glycerol, in the presence of a second selective reactionpromoter, which is capable of reacting lipoprotein lipase selectivelywith triglycerides contained in very low density lipoprotein,intermediate density lipoprotein, low density lipoprotein and highdensity lipoprotein, to generate hydrogen peroxide or a reduced coenzymefrom the triglycerides contained in the very low density lipoprotein andthe intermediate density lipoprotein or in the very low densitylipoprotein; and

2′. measuring the hydrogen peroxide or reduced coenzyme generated by thereaction 1′.

(2) The method according to the above description (1), wherein thesecond selective reaction promoter is an ether or ester compound of apolyoxyalkylene.

(3) The method according to the above description (2), wherein m/n ratiois in the range of 1.1 to 1.2 where m is the average mole number of theadded polyoxyalkylene in its ether or ester compound which is used asthe first selective reaction promoter and n is the average mole numberof the added polyoxyalkylene in its ether or ester compound which isused as the second selective reaction promoter.

(4) The method according to the above description (3), wherein m is inthe range of 7.7 to 18 and n is in the range of 7 to 15.

(5) The method according to the above description (3), wherein m is inthe range of 11 to 12and n is 10.

(6) The method according to any one of the above descriptions (1) to(5), wherein the ether or ester compound of a polyoxyalkylene which isused as the first selective reaction promoter is at least one selectedfrom the group consisting of polyoxyalkylene straight-chain alkylethers, polyoxyalkylene branched-chain alkyl ethers, polyoxyalkylenestraight-chain alkylphenyl ethers, polyoxyalkylene branched-chainalkylphenyl ethers, polyoxyalkylene straight-chain fatty acid esters,polyoxyalkylene branched-chain fatty acid esters, polyoxyalkylenestraight-chain alkyl substituted benzoic acid esters and polyoxyalkylenebranched-chain alkyl substituted benzoic acid esters.

(7) The method according to any one of the above descriptions (1) to(6), wherein the second selective reaction promoter is at least oneether or ester compound of a polyoxyalkylene selected from the groupconsisting of polyoxyalkylene straight-chain alkyl ethers,polyoxyalkylene branched-chain alkyl ethers, polyoxyalkylenestraight-chain alkylphenyl ethers, polyoxyalkylene branched-chainalkylphenyl ethers, polyoxyalkylene straight-chain fatty acid esters,polyoxyalkylene branched-chain fatty acid esters, polyoxyalkylenestraight-chain alkyl substituted benzoic acid esters and polyoxyalkylenebranched-chain alkyl substituted benzoic acid esters.

(8) The method according to any one of the above descriptions (1) to(7), wherein the polyoxyalkylene is polyoxyethylene.

(9) The method according to any one of the above descriptions (1) to(8), wherein the first selective reaction promoter is polyoxyethylenenonylphenyl ether in which the average mole number of addedpolyoxyethylene m is in the range of 11 to 12 and the second selectivereaction promoter is polyoxyethylene nonylphenyl ether in which theaverage mole number of added polyoxyethylene n is 10.

(10) The method according to any one of the above descriptions (1) to(9), wherein the first step and/or the second step is carried out in thepresence of a reaction assistant.

(11) The method according to the above descriptions (10), wherein thereaction assistant is a polysaccharide or derivative thereof, apolyanion, a halogen ion, a metal ion, or lectin.

(12) The method according to any one of the above descriptions (1) to(11), wherein the activity of the lipoprotein lipase being present inthe first step depends on the concentration of a surfactant, while thatof the lipoprotein lipase being present in the second step hardlydepends on the concentration of a surfactant.

(13) A reagent for selective measurement of triglycerides contained invery low density lipoprotein and intermediate density lipoprotein or invery low density lipoprotein in a test sample, including

a first reagent that comprises: a first selective reaction promoter,which is an ether or ester compound of a polyoxyalkylene capable ofreacting lipoprotein lipase selectively with triglycerides contained inlow density lipoprotein and high density lipoprotein; lipoproteinlipase; enzymes which catalyze a series of reactions leading to thegeneration of hydrogen peroxide or a reduced coenzyme from glycerol; andan enzyme which catalyzes the reaction leading to the conversion ofhydrogen peroxide or a reduced coenzyme into another substance, and

a second reagent that comprises a second selective reaction promoter,which is capable of reacting lipoprotein lipase selectively withtriglycerides contained in very low density lipoprotein, intermediatedensity lipoprotein, low density lipoprotein and high densitylipoprotein.

(14) The reagent according to the above description (13), wherein thefirst reagent and/or the second reagent further comprises a substancewhich is involved in a reaction leading to the derivation of some signalfrom hydrogen peroxide or a reduced coenzyme.

(15) The reagent according to the above description (13) or (14),wherein the second selective reaction promoter is an ether or estercompound of a polyoxyalkylene.

(16) The reagent according to the above description (15), wherein m/nratio is in the range of 1.1 to 1.2 where m is the average mole numberof the added polyoxyalkylene in its ether or ester compound which isused as the first selective reaction promoter and n is the average molenumber of the added polyoxyalkylene in its ether or ester compound whichis used as the second selective reaction promoter.

(17) The reagent according to the above description (16), wherein m isin the range of 7.7 to 18 and n is in the range of 7 to 15.

(18) The reagent according to the above description (16), wherein m isin the range of 11 to 12 and n is 10.

(19) The reagent according to any one of the above descriptions (13) to(18), wherein the ether or ester compound of a polyoxyalkylene used asthe first selective reaction promoter is at least one selected from thegroup consisting of polyoxyalkylene straight-chain alkyl ethers,polyoxyalkylene branched-chain alkyl ethers, polyoxyalkylenestraight-chain alkylphenyl ethers, polyoxyalkylene branched-chainalkylphenyl ethers, polyoxyalkylene straight-chain fatty acid esters,polyoxyalkylene branched-chain fatty acid esters, polyoxyalkylenestraight-chain alkyl substituted benzoic acid esters and polyoxyalkylenebranched-chain alkyl substituted benzoic acid esters.

(20) The reagent according to any one of the above descriptions (13) to(19), wherein the second selective reaction promoter is at least oneether or ester compound of a polyoxyalkylene selected from the groupconsisting of polyoxyalkylene straight-chain alkyl ethers,polyoxyalkylene branched-chain alkyl ethers, polyoxyalkylenestraight-chain alkylphenyl ethers, polyoxyalkylene branched-chainalkylphenyl ethers, polyoxyalkylene straight-chain fatty acid esters,polyoxyalkylene branched-chain fatty acid esters, polyoxyalkylenestraight-chain alkyl substituted benzoic acid esters and polyoxyalkylenebranched-chain alkyl substituted benzoic acid esters.

(21) The reagent according to any one of the above descriptions (13) to(20), wherein the polyoxyalkylene is polyoxyethylene.

(22) The reagent according to any one of the above descriptions (13) to(21), wherein the first selective reaction promoter is polyoxyethylenenonylphenyl ether in which the average mole number of addedpolyoxyethylene m is in the range of 11 to 12 and the second selectivereaction promoter is polyoxyethylene nonylphenyl ether in which theaverage mole number of added polyoxyethylene n is 10.

(23) The reagent according to any one of the above descriptions (13) to(22), wherein the first reagent and/or the second reagent furthercomprises a reaction assistant.

(24) The reagent according to the above descriptions (23), wherein thereaction assistant is a polysaccharide or derivative thereof, apolyanion, a halogen ion, a metal ion, or lectin.

(25) The reagent according to any one of the above descriptions (13) to(24), wherein the activity of the lipoprotein lipase contained in thefirst reagent depends on the concentration of a surfactant, while thatof the lipoprotein lipase contained in the second reagent hardly dependson the concentration of a surfactant.

In the following this invention will be described in detail.

I. Measurement Method

I-1: General Introduction of Measurement Method

The method for selective measurement of triglycerides contained in verylow density lipoprotein and intermediate density lipoprotein or in verylow density lipoprotein in a test sample in accordance with thisinvention is carried out in the following first and second steps.

First Step:

1′. Expose and react a test sample to and with lipoprotein lipase andsome other enzymes, which catalyze a series of reactions leading to thegeneration of hydrogen peroxide or a reduced coenzyme from glycerol, inthe presence of a first selective reaction promoter, which is an etheror ester compound of a polyoxyalkylene capable of reacting lipoproteinlipase selectively with triglycerides contained in low densitylipoprotein and high density lipoprotein, to generate hydrogen peroxideor a reduced coenzyme from the triglycerides contained in the lowdensity lipoprotein and the high density lipoprotein in the test sample.

2′. React the hydrogen peroxide or reduced coenzyme generated by thereaction of the above description 1′ with an enzyme which catalyzes thereaction leading to the conversion of hydrogen peroxide or a reducedcoenzyme into another substance.

3′. Eliminate the triglycerides contained in the low density lipoproteinand the high density lipoprotein by the above reactions 1′ and 2′.

Second Step:

1′. Subsequently after the first step, react the test sample withlipoprotein lipase and some other enzymes, which catalyze a series ofreactions leading to the generation of hydrogen peroxide or a reducedcoenzyme from glycerol, in the presence of a second selective reactionpromoter, which is capable of reacting lipoprotein lipase selectivelywith triglycerides contained in very low density lipoprotein,intermediate density lipoprotein, low density lipoprotein and highdensity lipoprotein, to generate hydrogen peroxide or a reduced coenzymefrom the triglycerides contained in the very low density lipoprotein andthe intermediate density lipoprotein or in the very low densitylipoprotein.

2′. Measure the hydrogen peroxide or reduced coenzyme generated by theabove reaction 1′.

These operating procedures eliminate triglycerides contained in highdensity lipoprotein and low density lipoprotein in a test sample andinhibit triglycerides contained in chylomicron in the test sample frombeing involved in the measurement reactions, whereby they derivehydrogen peroxide or a reduced coenzyme exclusively from triglyceridescontained in very low density lipoprotein and intermediate densitylipoprotein or in very low density lipoprotein and enable the selectivemeasurement of such triglycerides.

I-2: First Step

The first step of the measurement method in accordance with thisinvention involves the following reactions:

1′. React lipoprotein lipase selectively with triglycerides contained inlow density lipoprotein and high density lipoprotein in a test sample byexposing the test sample to lipoprotein lipase and some other enzymes,which catalyze a series of reactions leading to the generation ofhydrogen peroxide or a reduced coenzyme from glycerol, in the presenceof a first selective reaction promoter, which is an ether or estercompound of a polyoxyalkylene capable of reacting lipoprotein lipaseselectively with triglycerides contained in low density lipoprotein andhigh density lipoprotein, to generate glycerol.

React the generated glycerol with enzymes that catalyze a series ofreactions leading to the generation of hydrogen peroxide or a reducedcoenzyme to derive hydrogen peroxide or a reduced coenzyme from theglycerol.

2′. React the hydrogen peroxide or reduced coenzyme generated by theabove reaction 1′ with an enzyme which catalyzes a reaction leading tothe conversion of hydrogen peroxide or a reduced coenzyme into anothersubstance so that neither hydrogen peroxide nor reduced coenzyme ispresent in the reaction system.

3′. Convert the triglycerides contained in the low density lipoproteinand the high density lipoprotein into another substances by the abovereactions 1′ and 2′ to eliminate the triglycerides from the reactionsystem.

I-3: Second Step

The second step of the measurement method in accordance with thisinvention involves the following reactions.

1′. Subsequently after the first step, expose the test sample to asecond selective reaction promoter, which is capable of reactinglipoprotein lipase selectively with triglycerides contained in very lowdensity lipoprotein, intermediate density lipoprotein, low densitylipoprotein and high density lipoprotein, in the reaction system of thefirst step.

Then, react the test sample with lipoprotein lipase and some otherenzymes, which catalyze a series of reactions leading to the generationof hydrogen peroxide or a reduced coenzyme from glycerol, in thepresence of the second selective reaction promoter. Since thetriglycerides contained in the low density lipoprotein and the highdensity lipoprotein have been already eliminated in the first step, onlythe triglycerides contained in the very low density lipoprotein andintermediate density lipoprotein or in the very low density lipoproteinreact with lipoprotein lipase to generate glycerol.

React the generated glycerol with enzymes which catalyze a series ofreaction leading to the generation of hydrogen peroxide or a reducedcoenzyme from glycerol to derive hydrogen peroxide or a reduced coenzymefrom the glycerol.

2′. Measure the hydrogen peroxide or reduced coenzyme generated by theabove reaction 1′ directly or using some signal derived from thehydrogen peroxide or reduced coenzyme.

I-4. Selective Reaction Promoters

I-4-1: First Selective Reaction Promoter

The first selective reaction promoter used in this invention is asubstance which is capable of reacting lipoprotein lipase selectivelywith triglycerides contained in low density lipoprotein and high densitylipoprotein.

In other words, in the presence of the first selective reactionpromoter, the reaction of lipoprotein lipase which leads to thegeneration of glycerol from triglycerides contained in lipoproteinsoccurs and progresses selectively in the triglycerides contained in lowdensity lipoprotein and high density lipoprotein.

As the first selective reaction promoter, an ether compound or estercompound of a polyoxyalkylene is used.

Examples of polyoxyalkylene ether compounds suitably used as aboveinclude polyoxyalkylene straight-chain alkyl ethers (e.g.polyoxyethylene octyl ether, polyoxyethylene nonyl ether,polyoxyethylene lauryl ether, polyoxyethylene cetyl ether,polyoxyethylene stearyl ether, polyoxyethylene oleyl ether,polyoxyethylene behenyl ether), polyoxyalkylene branched-chain alkylethers, polyoxyalkylene straight-chain alkylphenyl ethers (e.g.polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenyl ether,polyoxyethylene laurylphenyl ether, polyoxyethylene stearylphenylether), and polyoxyalkylene branched-chain alkylphenyl ethers.

Examples of polyoxyalkylene ester compounds suitably used as aboveinclude polyoxyalkylene straight-chain fatty acid esters (e.g.polyoxyalkylene laurate, polyoxyalkylene stearate, polyoxyalkyleneoleate, polyoxyalkylene coconut oil fatty acid ester), polyoxyalkylenebranched-chain fatty acid esters, polyoxyalkylene straight-chain alkylsubstituted benzoic acid esters (e.g. polyoxyethylene octylbenzoate,polyoxyethylene nonylbenzoate, polyoxyethylene laurylbenzoate,polyoxyethylene stearylbenzoate,) and polyoxyalkylene branched-chainalkyl substituted benzoic acid esters.

Particularly suitably used are polyoxyalkylene straight-chainalkylphenyl ethers and polyoxyalkylene branched-chain alkylphenylethers.

The straight-chain alkyl groups and branched-chain alkyl groups in theabove described polyoxyalkylene straight-chain alkyl ethers,polyoxyalkylene branched-chain alkyl ethers, polyoxyalkylenestraight-chain alkylphenyl ethers, polyoxyalkylene branched-chainalkylphenyl ethers, polyoxyalkylene straight-chain alkyl substitutedbenzoic acid esters and polyoxyalkylene branched-chain alkyl substitutedbenzoic acid esters as well as the straight-chain fatty acids andbranched-chain fatty acids in the above described polyoxyalkylenestraight-chain fatty acid esters and polyoxyalkylene branched-chainfatty acid esters are not limited to any specific ones; however, thoseof C₇₋₃₀ are preferably used.

The polyoxyalkylene group of the above described polyoxyalkylene etheror ester compounds is preferably a polyoxyethylene group.

Either one kind or two or more kinds of first selective reactionpromoters may be selected from among the above described ones and used(present (contained)) in the reaction system.

The concentration of a first selective reaction promoter(s) present(contained) in the reaction system cannot be generalized, because itvaries depending on the kind of the first selective reaction promoter(s)selected, the kind and origin of the enzymes which catalyze the seriesof reactions leading to the generation of hydrogen peroxide or a reducedcoenzyme from triglycerides, the concentration of the triglyceridescontained in lipoproteins in a test sample or, when measurement is madeusing a two-reagent system, the mixing ratio of the first reagent to thesecond reagent. Accordingly, the first selective reaction promoter maybe present (contained) in the reaction system at a concentrationsuitable for such conditions. Generally a first selective reactionpromoter(s) is present (contained) in the reaction system at aconcentration of 0.005 to 5%, preferably 0.01 to 2% and particularlypreferably 0.05 to 1%.

I-4-2: Second Selective Reaction Promoter

The second selective reaction promoter used in this invention is asubstance which is capable of reacting lipoprotein lipase selectivelywith triglycerides contained in very low density lipoprotein,intermediate density lipoprotein, low density lipoprotein and highdensity lipoprotein.

In other words, in the presence of the second selective reactionpromoter, the reaction of lipoprotein lipase which leads to thegeneration of glycerol from triglycerides contained in lipoproteinsoccurs and progresses selectively in the triglycerides contained in verylow density lipoprotein, intermediate density lipoprotein, low densitylipoprotein and high density lipoprotein.

As the second selective reaction promoter, an ether compound or estercompound of a polyoxyalkylene is suitably used.

Examples of polyoxyalkylene ether compounds suitably used as aboveinclude polyoxyalkylene straight-chain alkyl ethers (e.g.polyoxyethylene octyl ether, polyoxyethylene nonyl ether,polyoxyethylene lauryl ether, polyoxyethylene cetyl ether,polyoxyethylene stearyl ether, polyoxyethylene oleyl ether,polyoxyethylene behenyl ether), polyoxyalkylene branched-chain alkylethers, polyoxyalkylene straight-chain alkylphenyl ethers (e.g.polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenyl ether,polyoxyethylene laurylphenyl ether, polyoxyethylene stearylphenylether), and polyoxyalkylene branched-chain alkylphenyl ethers.

Examples of polyoxyalkylene ester compounds suitably used as aboveinclude polyoxyalkylene straight-chain fatty acid esters (e.g.polyoxyalkylene laurate, polyoxyalkylene stearate, polyoxyalkyleneoleate, polyoxyalkylene coconut oil fatty acid ester), polyoxyalkylenebranched-chain fatty acid esters, polyoxyalkylene straight-chain alkylsubstituted benzoic acid esters (e.g. polyoxyethylene octylbenzoate,polyoxyethylene nonylbenzoate, polyoxyethylene laurylbenzoate,polyoxyethylene stearylbenzoate,) and polyoxyalkylene branched-chainalkyl substituted benzoic acid esters.

Particularly suitably used are polyoxyalkylene straight-chainalkylphenyl ethers and polyoxyalkylene branched-chain alkylphenylethers.

The straight-chain alkyl groups and branched-chain alkyl groups in theabove described polyoxyalkylene straight-chain alkyl ethers,polyoxyalkylene branched-chain alkyl ethers, polyoxyalkylenestraight-chain alkylphenyl ethers, polyoxyalkylene branched-chainalkylphenyl ethers, polyoxyalkylene straight-chain alkyl substitutedbenzoic acid esters and polyoxyalkylene branched-chain alkyl substitutedbenzoic acid esters as well as the straight-chain fatty acids andbranched-chain fatty acids in the above described polyoxyalkylenestraight-chain fatty acid esters and polyoxyalkylene branched-chainfatty acid esters are not limited to any specific ones; however, thoseof C₇₋₃₀ are preferably used.

The polyoxyalkylene group of the above described polyoxyalkylene etheror ester compounds is preferably a polyoxyethylene group.

Either one kind or two or more kinds of second selective reactionpromoters may be selected from among the above described ones and used(present (contained)) in the reaction system.

The concentration of the second selective reaction promoter(s) present(contained) in the reaction system cannot be generalized, because itvaries depending on the kind of the second selective reactionpromoter(s) selected, the kind and source of the enzymes which catalyzethe series of reactions leading to the generation of hydrogen peroxideor a reduced coenzyme from triglycerides, the concentration of thetriglycerides contained in lipoproteins in a test sample or, whenmeasurement is made using a two-reagent system, the mixing ratio of thefirst reagent to the second reagent. Accordingly, the second selectivereaction promoter may be present (contained) in the reaction system at aconcentration suitable for such conditions. The concentration of thesecond selective reaction promoter(s) present (contained) in thereaction system is usually 0.005 to 5%, preferably 0.01 to 2% andparticularly preferably 0.05 to 1%.

In this invention, however, care should be taken not to allow thecomposition and concentration of the second selective reaction promoterin the second step and those of the first selective reaction promoter inthe first step to be completely the same.

I-4-3: Average Mole Number of Added Polyoxyalkylene in SelectiveReaction Promoters

Where the average mole number of the added polyoxyalkylene in apolyoxyalkylene ether compound or polyoxyalkylene ester compound as thefirst selective reaction promoter is m and that of the addedpolyoxyalkylene in a polyoxyalkylene ether compound or polyoxyalkyleneester compound as the second selective reaction promoter is n, it ispreferable that the m/n ratio is in the range of 1.1 to 1.2.

It is more preferable that the m/n ratio is in the range of 1.1 to 1.2,m is in the range of 7.7 to 18 and n is in the range of 7 to 15. And itis particularly preferable that the m/n ratio is in the range of 1.1 to1.2, m is in the range of 11 to 12 and n is 10.

It is also particularly preferable that the above described firstselective reaction promoter is polyoxyethylene nonylphenyl ether inwhich the average mole number of the added polyoxyethylene m is in therange of 11 to 12 and the above described second selective reactionpromoter is polyoxyethylene nonylphenyl ether in which the average molenumber of the added polyoxyethylene n is 10.

The larger the value m becomes, the more selectively triglyceridescontained in very low density lipoprotein can be measured than thosecontained in intermediate density lipoprotein.

The smaller the value m becomes, the more selectively triglyceridescontained in intermediate density lipoprotein can be measured than thosecontained in very low density lipoprotein.

And the larger the m/n ratio becomes, the more selectively triglyceridescontained in very low density lipoprotein can be measured than thosecontained in intermediate density lipoprotein.

Thus, the selectivity can be varied depending on the objective of themeasurement by properly setting the value m or the m/n ratio.

I-5: Reactions Leading to the Generation of Hydrogen Peroxide or aReduced Coenzyme from Triglycerides

I-5-1: Reactions Leading to the Generation of Hydrogen Peroxide or aReduced Coenzyme from Triglycerides

In the measurement method of this invention, the reactions in the firstand second steps which lead to the generation of hydrogen peroxide or areduced coenzyme from triglycerides include: a reaction catalyzed bylipoprotein lipase; and a series of reactions leading to the generationof hydrogen peroxide and a reduced coenzyme from glycerol.

I-5-2: Reaction Catalyzed by Lipoprotein Lipase

The reaction catalyzed by lipoprotein lipase in this invention is areaction leading to the hydrolysis of triglycerides contained inlipoproteins in a test sample into one molecule of glycerol and threemolecules of fatty acid by exposing the triglycerides to lipoproteinlipase.

Any lipoprotein lipase can be used as long as it catalyzes the reactionleading to the hydrolysis of triglycerides into one molecule of glyceroland three molecules of fatty acid.

The lipoprotein lipase used in this invention may be derived frommicroorganisms such as bacteria or fungi, derived from animals such ashuman beings, pigs or cows, derived from plants, or prepared by therecombinant DNA technique.

The activity level of lipoprotein lipase present (contained) in thereaction system cannot be generalized, because it varies depending onthe origin of the lipoprotein lipase, the kind of the first or secondselective reaction promoter or, when measurement is made using atwo-reagent system, the mixing ratio of the first reagent to the secondreagent. Lipoprotein lipase may therefore be used at a level suitablefor such conditions.

Generally preferably lipoprotein lipase is present (contained) in thereaction system at an activity level of 1 to 10,000,000 units/l and morepreferably at an activity level of 100 to 1,000,000 units/l.

Normally, the activity level of an enzyme varies depending on the methodemployed for its measurement, and besides, even if the same method isemployed for the same type of enzyme, the activity level obtained variesdepending on the origin or the purity of the enzyme. Accordingly, evenif the activity level of the lipoprotein lipase used is outside theabove described range, the lipoprotein lipase can sometimes provideadvantageous effects of this invention.

Preferably, the activity of the lipoprotein lipase present in the firststep of the measurement method of this invention depends on theconcentration of a surfactant, while that of the lipoprotein lipasepresent in the second step hardly depends on the concentration of asurfactant.

The “lipoprotein lipase whose activity depends on the concentration of asurfactant” means lipoprotein lipase whose activity increases with theincrease in the concentration of a surfactant.

The “lipoprotein lipase whose activity hardly depends on theconcentration of a surfactant” means lipoprotein lipase whose activityrapidly increases with the increase in the concentration of a surfactantand reaches a certain level, and from that point on, hardly changes evenif the concentration of the surfactant increases.

Accordingly, discrimination between the above two types of lipoproteinlipase can be made by measuring the degree and pattern of increase inenzyme activity when increasing the concentration of a surfactantpresent in the reaction system.

One example of methods for this discrimination is shown in “Experimentalexample” described later.

For example, when dividing the measured value obtained in accordancewith the method described in “Experimental example” by the measuredvalue obtained when the reaction system contains lipoprotein lipasewhose activity hardly depends on the concentration of a surfactant (e.g.LPL or LPL-311), if the quotient is 0.5 or less, the lipoprotein lipaseis judged to be lipoprotein lipase whose activity depends on theconcentration of a surfactant, whereas if the quotient is more than 0.5,the lipoprotein lipase is judged to be lipoprotein lipase whose activityhardly depends on the concentration of a surfactant.

Examples of the above described surfactants include nonionicsurfactants, anionic surfactants, cationic surfactants and amphotericsurfactants. Particularly preferable are nonionic surfactants.

Examples of such nonionic surfactants include ether compounds or estercompounds of polyoxyalkylene. Particularly preferable arepolyoxyalkylene branched-chain alkyl ether and polyoxyalkylenealkylphenyl formaldehyde condensates.

Examples of lipoprotein lipase whose activity depends on theconcentration of a surfactant include “LP-BP” (Asahi Kasei Corp.) and“LPL-314” (Toyobo Co., Ltd.).

Examples of lipoprotein lipase whose activity hardly depends on theconcentration of a surfactant include “LPL” (Asahi Kasei Corp.) and“LPL-311” (Toyobo Co., Ltd.).

I-5-3: A Series of Reactions Leading to the Generation of HydrogenPeroxide or Reduced Coenzyme from Glycerol

The series of reactions leading to the generation of hydrogen peroxideor reduced coenzyme in this invention may be any reaction as long as itenables the generation of hydrogen peroxide or a reduced coenzyme fromglycerol as a hydrolysis product by lipoprotein lipase. It may consistof a single reaction or a plurality of reactions.

Examples of reduced coenzymes include nicotinamide adenine dinucleotide(reduced form) [NADH (reduced)] and nicotinamide adenine dinucleotidephosphate (reduced form) [NADPH (reduced)].

One example of such series of reactions leading to the generation ofhydrogen peroxide or a reduced coenzyme is a series of reactions thatconverts glycerol and adenosine triphosphate (ATP) intoglycerol-3-phosphate and adenosine diphosphate (ADP) by the catalyticaction of glycerol kinase and further converts the resultantglycerol-3-phosphate into dihydroxyacetone-3-phosphate, while generatinghydrogen peroxide, by the catalytic action of glycerol-3-phosphateoxidase.

Another example of such series of reactions is a series of reactionsthat converts glycerol and adenosine triphosphate (ATP) intoglycerol-3-phosphate and adenosine diphosphate (ADP) by the catalyticaction of glycerol kinase and further converts the resultantglycerol-3-phosphate into dihydroxyacetone-3-phosphate, while generatingnicotinamide adenine dinucleotide (reduced form) [NADH], in the presenceof nicotinamide adenine dinucleotide (oxidized form) [NAD⁺] by thecatalytic action of glycerol-3-phosphate dehydrogenase.

In the method of this invention, the enzymes that catalyze a series ofreaction leading to the generation of hydrogen peroxide or a reducedcoenzyme from glycerol may be any enzymes as long as they can catalyzethe series of reaction leading to the generation of hydrogen peroxide ora reduced coenzyme from glycerol. Examples of such enzymes include: acombination of glycerol kinase and glycerol-3-phosphate oxidase, and acombination of glycerol kinase and glycerol-3-phosphate dehydrogenase.

The enzymes used in this invention may be derived from microorganismssuch as bacteria or fungi, derived from animals such as human beings,pigs or cows, derived from plants, or prepared by the recombinant DNAtechnique.

The activity levels of the enzymes present (contained) in the reactionsystem cannot be generalized, because they vary depending on the kindand origin of the enzymes, the kind of the first or second selectivereaction promoter or, when measurement is made using a two-reagentsystem, the mixing ratio of the first reagent to the second reagent. Theenzymes may therefore be used at a level suitable for such conditions.

Generally preferably glycerol kinase is present (contained) in thereaction system at an activity level of 0.01 to 500,000 units/i and morepreferably at an activity level of 10 to 10,000 units/l.

Also, generally preferably glycerol-3-phosphate oxidase is present(contained) in the reaction system at an activity level of 1 to 500,000units/I and more preferably at an activity level of 100 to 50,000units/l.

Normally, the activity level of an enzyme varies depending on the methodemployed for its measurement, and besides, even if the same method isemployed for the same type of enzyme, the activity level obtained variesdepending on the origin or the purity of the enzyme. Accordingly, evenif the activity levels of the enzymes used are outside the abovedescribed range, the enzymes can sometimes provide advantageous effectsof this invention.

The catalytic reaction by glycerol kinase requires adenosinetriphosphate (ATP) and magnesium ion. Accordingly, when using glycerolkinase, adenosine triphosphate and magnesium ion are added to (orcontained in) the reaction systems of the first and the second steps.

Adenosine triphosphate used may be in the form of a free acid or a salt.The concentration of adenosine triphosphate or the salt thereof present(contained) in the reaction system is generally preferably 0.001 to 50g/l and particularly preferably 0.01 to 10 g/l.

The magnesium ion used in the reaction may be in the form of a salt witha halogen ion or an organic acid. It is generally preferably present(contained) in the reaction system at a concentration of 0.001 to 100 mMand particularly preferably at a concentration of 0.01 to 50 mM.

The catalytic reaction by glycerol-3-phosphate dehydrogenase requires anoxidized coenzyme such as nicotinamide adenine dinucleotide (oxidizedform) [NAD⁺] or nicotinamide adenine dinucleotide phosphate (oxidizedform) [NADP⁺ (oxidized)]. Accordingly, when using glycerol-3-phosphatedehydrogenase, the oxidized coenzyme is added to (or contained in) thereaction systems of the first and the second steps.

I-6: Reaction Leading to the Conversion of Hydrogen Peroxide or ReducedCoenzyme into another Substance

In the first step of the measurement method of this invention, hydrogenperoxide or a reduced coenzyme is generated from triglycerides containedin low density lipoprotein and high density lipoprotein in the presenceof a first selective reaction promoter and the generated hydrogenperoxide or reduced coenzyme is converted into another substance.

This series of reactions leads to the elimination of triglyceridescontained in low density lipoprotein and high density lipoprotein.

The reaction leading to the conversion of hydrogen peroxide or a reducedcoenzyme into another substance may be any reaction as long as it canconvert hydrogen peroxide or a reduced coenzyme into another substance.The reaction may consist of a single reaction or a plurality ofreactions.

I-6-1: Reaction Leading to the Conversion of Hydrogen Peroxide intoanother Substance

I-6-1-1: Reaction by Catalase

One example of reactions leading to the conversion of hydrogen peroxideinto another substance is a catalytic reaction by catalase that degradestwo molecules of hydrogen peroxide to two molecules of water and onemolecule of oxygen.

Any type of catalase may be used as long as it catalyzes the reactionleading to the degradation of two molecules of hydrogen peroxide to twomolecules of water and one molecule of oxygen.

The catalase used in the reaction may be derived from microorganismssuch as bacteria or fungi, derived from animals such as human beings,pigs or cows, derived from plants, or prepared by the recombinant DNAtechnique.

The activity level of catalase present (contained) in the reactionsystem cannot be generalized, because it varies depending on the originof the catalase or, when measurement is made using a two-reagent system,the mixing ratio of the first reagent to the second reagent. Catalasemay therefore be used at a level suitable for such conditions.

Generally preferably catalase is present (contained) in the reactionsystem at an activity level of 100 units/l or more.

Normally, the activity level of an enzyme varies depending on the methodemployed for its measurement, and besides, even if the same method isemployed for the same type of enzyme, the activity level obtained variesdepending on the origin or the purity of the enzyme. Accordingly, evenif the activity level of the catalase used is outside the abovedescribed range, the catalase can sometimes provide advantageous effectsof this invention.

After eliminating the generated hydrogen peroxide with catalase in thefirst step, it is necessary in the second step to deactivate thecatalase so that hydrogen peroxide generated in the second step shouldnot be eliminated (degraded) by the catalase.

This can be accomplished by addition of a substance that deactivatecatalase, such as sodium azide, to the reaction system in the secondstep.

I-6-1-2: Reaction by Peroxidase

Another example of reactions leading to the conversion of hydrogenperoxide into another substance is a catalytic reaction by peroxidase(POD) that produces an oxidized substance from hydrogen peroxide and anoxidizable substance (e.g. a chromogen in a Trinder reaction system).

Any type of POD may be used as long as it catalyzes the reaction leadingto the production of an oxidized substance from hydrogen peroxide and anoxidizable substance, and hence the consumption of hydrogen peroxide.

The POD used in the reaction may be derived from microorganisms such asbacteria or fungi, derived from animals such as human beings, pigs orcows, derived from plants such as horseradish, or prepared by therecombinant DNA technique.

The activity level of POD present (contained) in the reaction systemcannot be generalized, because it varies depending on the origin of thePOD or, when measurement is made using a two-reagent system, the mixingratio of the first reagent to the second reagent. POD may therefore beadded to (or contained in) the reaction system in the first step at alevel suitable for such conditions.

Generally preferably POD is present (contained) in the reaction systemat an activity level of 30 units/l or more.

Normally, the activity level of an enzyme varies depending on the methodemployed for its measurement, and besides, even if the same method isemployed for the same type of enzyme, the activity level obtained variesdepending on the origin or the purity of the enzyme. Accordingly, evenif the activity level of the POD used is outside the above describedrange, the POD can sometimes provide advantageous effects of thisinvention.

Examples of substances to be oxidized include chromogens in a Trinderreaction system.

Examples of chromogens in a Trinder reaction system include4-aminoantipyrine, phenol or derivatives thereof, or anilinederivatives.

If both 4-aminoantipyrine and phenol or derivative thereof or both4-aminoantipyrine and an aniline derivative exist together in thereaction system of the first step, color is developed by the generatedhydrogen peroxide and POD; therefore, preferably either4-aminoantipyrine or phenol or derivative thereof alone, or either4-aminoantipyrine or an aniline derivative alone is present (contained)in the reaction system.

The details of chromogens in a Trinder reaction system will be describedlater in I-7-1: Hydrogen peroxide (I-7: Measurement of hydrogen peroxideor reduced coenzyme).

I-6-2: Reaction Leading to the Conversion of Reduced Coenzyme intoanother Substance

One example of reactions leading to the conversion of a reduced coenzymeinto another substance is a catalytic reaction by a dehydrogenase usingthe reduced coenzyme as a coenzyme that converts the reduced coenzymeinto an oxidized coenzyme.

Any type of dehydrogenase may be used as long as it catalyzes thereaction leading to the conversion of the reduced coenzyme into anoxidized coenzyme using the reduced coenzyme as a coenzyme. Examples ofsuch types of dehydrogenase include lactate dehydrogenase, malatedehydrogenase and isocitrate dehydrogenase.

The dehydrogenase used in the reaction may be derived frommicroorganisms such as bacteria or fungi, derived from animals such ashuman beings, pigs or cows, derived from plants, or prepared by therecombinant DNA technique.

The activity level of dehydrogenase present (contained) in the reactionsystem cannot be generalized, because it varies depending on the originof the dehydrogenase or, when measurement is made using a two-reagentsystem, the mixing ratio of the first reagent to the second reagent.Dehydrogenase may therefore be added to the reaction system of the firststep at a level suitable for such conditions.

I-7: Measurement of Hydrogen Peroxide or Reduced Coenzyme

In the second step of the measurement method of this invention, hydrogenperoxide or a reduced coenzyme is generated from triglycerides containedin very low density lipoprotein and intermediate density lipoprotein, orin very low density lipoprotein in the presence of a second selectivereaction promoter and the generated hydrogen peroxide or reducedcoenzyme is measured.

The measurement of triglycerides contained in very low densitylipoprotein and intermediate density lipoprotein, or in very low densitylipoprotein in a test sample is made through the series of reactions.

The method for measuring the hydrogen peroxide or reduced coenzymegenerated in the second step may be any method as long as it enables themeasurement of the amount or the presence of hydrogen peroxide or areduced coenzyme generated through the above described series ofreactions that leads to the generation of hydrogen peroxide or a reducedcoenzyme from glycerol.

One example of such methods is a method in which some signal is derivedfrom the generated hydrogen peroxide or reduced coenzyme.

I-7-1: Hydrogen Peroxide

To measure hydrogen peroxide, a hydrogen peroxide electrode or the likemay be used to directly measure hydrogen peroxide or some signal may bederived from hydrogen peroxide to measure the signal.

One example of methods in which some signal is derived from hydrogenperoxide is a method utilizing a Trinder reaction system in which achromogen is oxidized to generate a dye in the presence of POD and theabsorbance of the generated dye is measured.

The POD used in the reaction may be derived from microorganisms such asbacteria or fungi, derived from animals such as human beings, pigs orcows, derived from plants such as horseradish, or prepared by therecombinant DNA technique.

Generally preferably the activity level of the POD present (orcontained) in the reaction system is 30 units/l or more.

Examples of chromogens in a Trinder reaction system include thecombination of 4-aminoantipyrine and phenol or derivative thereof, orthe combination of 4-aminoantipyrine and an aniline derivative.

Generally preferably 4-aminoantipyrine is present (or contained) in thereaction system at a concentration of 0.001 to 50 g/l and particularlypreferably at a concentration of 0.01 to 10 g/l.

Examples of phenol derivatives include 4-chlorophenol,2,4-dichlorophenol, 2,4-dibromophenol, 2,4,6-trichlorophenol, and thesalts thereof.

Examples of aniline derivatives includeN-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS),N-(3-sulfopropyl)-3,5-dimethoxyaniline (HDAPS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline (DAOS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline(FDAOS), N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxy-4-fluoroaniline(FDAPS), N-(2-carboxyethyl)-N-ethyl-3,5-dimethoxyaniline (CEDB),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (ADOS),N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline (ADPS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline (ALOS),N-ethyl-N-(3-sulfopropyl)aniline (ALPS), N-(3-sulfopropyl)aniline(HALPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS),N-ethyl-N-(3-sulfopropyl)-3,5-dimethylaniline (MAPS),N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline (TOOS),N-(2-carboxyethyl)-N-ethyl-3-methylaniline (CEMB),N-(2-carboxyethyl)-N-ethyl-3-methoxyaniline (CEMO) and the saltsthereof.

Generally preferably the above described phenol or derivative thereof,or aniline derivative is present (or contained) in the reaction systemat a concentration of 0.001 to 50 g/l and particularly preferably at aconcentration of 0.01 to 10 g/l.

I-7-2: Reduced Coenzyme

Measurement of a reduced coenzyme may be made by, for example, measuringthe absorbance of the reduced coenzyme itself, for example, at 340 nm orby deriving some other signal from the reduced coenzyme, followed bymeasuring the signal.

Examples of methods for deriving some signal from the reduced enzymeinclude a method utilizing a reaction that involves the reduction of atetrazolium salt or the like in the presence of diaphorase,1-methoxy-phenazine methosulfate or the like to produce a dye andmeasuring the dye.

I-8: Reaction Assistant

I-8-1: Reaction Assistant

In the measurement method of this invention, a reaction assistant may bepresent (or contained) in the reaction system of the first and/or thesecond step.

The presence (or addition) of a reaction assistant enhances theselective-reaction-promotion action of the first selective reactionpromoter and/or the second selective reaction promoter.

Concrete examples of reaction assistants include polysaccharides orderivatives thereof, polyanions, halogen ions, metal ions, lectin andthe like.

These reaction assistants may be present (or contained) in the reactionsystem in combination.

I-8-2: Polysaccharides or Derivatives Thereof

Examples of polysaccharides used in the measurement method of thisinvention include saccharides produced by dehydration condensation ofseveral or more monosaccharides. Concrete examples are cyclodextrin(CD), dextran, heparin and the like.

Examples of the derivatives of polysaccharides include those produced bysubstituting hydrogen or a functional group such as hydroxyl group ofthe polysaccharides, with: hydroxypropyl group; hydroxybutyl group;glucosyl group; maltosyl group; diethylaminoethyl group; an aliphatichydrocarbon group such as methyl or ethylidene; an alicyclic hydrocarbongroup such as cyclopropyl; an aromatic hydrocarbon group such as phenyl,benzyl or benzylidene; an ether group such as methoxy or phenoxy; anester group such as acetoxy or benzoyloxy; an acyl group such as acetyl,propionyl or benzoyl; a sulfhydryl group; a sulfo group; a sulfonylgroup, a carboxyl group; an amino group; an imino group; a nitryl group;a carbonyl group; an oxo group; a hydroxyl group; or a nitro group.

Examples of the derivatives of polysaccharides also include crosslinkedproducts of the above described polysaccharides or substitutedpolysaccharides.

Examples of the cyclodextrin used as the reaction assistant includeα-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD).

Examples of the cyclodextrin derivatives include: those produced bysubstituting a hydroxyl group of α-CD, β-CD or γ-CD with a group such ashydroxypropyl, hydroxybutyl, glucosyl, maltosyl, benzyl or sulfonyl; andcrosslinked products of the above described cyclodextrin or derivativesthereof.

Of the above described types of cyclodextrin, β-CD or γ-CD is preferablyused. Of the above described cyclodextrin derivatives, those produced bysubstituting a hydroxyl group of β-CD or γ-CD with a group as describedabove or crosslinked products of β-CD or γ-CD are preferably used.

When using dextran sulfate or derivative thereof, one whose molecularweight is in the range of 1,000 to 5,000,000 is preferably used and onewhose molecular weight is in the range of 5,000 to 1,000,000 isparticularly preferably used.

The concentration of polysaccharide or derivative thereof present(contained) in the reaction system cannot be generalized, because itvaries depending on the kind of the polysaccharide or derivativeselected, the kind and concentration of the first and/or secondselective reaction promoter, the kind and origin of the enzymes whichcatalyze the series of reactions leading to the generation of hydrogenperoxide or a reduced coenzyme from triglycerides, the concentration oftriglycerides contained in lipoproteins in a test sample or, whenmeasurement is made using a two-reagent system, the mixing ratio of thefirst reagent to the second reagent. Accordingly, polysaccharide orderivative thereof may be added (or contained) in the reaction system ata concentration suitable for such conditions. Generally polysaccharideor derivative thereof may be present (contained) in the reaction systemat a concentration of 0.005 to 5%, preferably at a concentration of 0.08to 2% and particularly preferably 0.01 to 1%.

I-8-3: Other Reaction Assistants

Examples of polyanions used as the reaction assistants includephosphotungstate or the like.

Examples of halogen ions include a chloride ion or the like.

Examples of metal ions include divalent metal ions such as copper ionand manganese ion, or the like.

Examples of lectin used as the reaction assistants include lentil lectinor the like.

The concentration of a reaction assistant other than polysaccharide orderivative thereof present (contained) in the reaction system cannot begeneralized, because it varies depending on the kind of the reactionassistant selected, the kind and concentration of the first and/orsecond selective reaction promoter, the kind and origin of the enzymeswhich catalyze the series of reactions leading to the generation ofhydrogen peroxide or a reduced coenzyme from triglycerides, theconcentration of triglycerides contained in lipoproteins in a testsample or, when measurement is made using a two-reagent system, themixing ratio of the first reagent to the second reagent. Accordingly, areaction assistant other than polysaccharide or derivative thereof maybe added (or contained) in the reaction system at a concentrationsuitable for such conditions.

I-9: Other Substances

In the measurement method of this invention, additional substances mayalso be added to the reaction system, depending on the situation.Examples of additional substances include: buffers; enzymes other thanthe above described ones; substrates for the enzymes; coenzymes otherthan the above described ones; ions or salts of alkaline metals,alkaline earth metals or the like; chelators; proteins such as albumin;sodium azide, antibiotics or preservatives such as a syntheticantibacterial agent; stabilizers such as saccharides or polymercompounds; activators; substances involved in the elimination orinhibition of interfering substances contained in a test sample, such asascorbate oxidase; excipients; and other reagent components.

Preferably the pH values of the reaction system in the first and secondsteps of the measurement method of this invention are in the range of 5to 10 and more preferably in the range of 5.5 to 9.0.

Accordingly, it is preferable the reaction system in the first andsecond steps contains a buffer that allows the pH of the reaction systemto be in the above described range.

Examples of such buffers include MES, Bis-Tris, Bis-Tris propane, ADA,PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO,EPPS, Tricine, Bicine, TAPS, CHES, phosphoric acid, phosphate, boricacid, borate, glycine, glycylglycine, imidazole, andtris(hydroxymethyl)aminomethane[Tris].

I-10: Test Samples

In the measurement method of this invention, a test sample may be anytest sample as long as it is suspected to include triglyceridescontained in very low density lipoprotein and intermediate densitylipoprotein, or in very low density protein and intended to be used forthe measurement of triglycerides contained in very low densitylipoprotein and intermediate density lipoprotein, or in very low densityprotein.

Examples of such test samples include: body fluids such as human oranimal blood, serum and plasma; extracted fluids from human or animalorgans or muscles; extracted fluids from human or animal feces;extracted fluids from cells and bacterial cells; and extracted fluidsfrom plants.

I-11: Measurement Operations

The measurement method of this invention is to selectively measuretriglycerides contained in very low density lipoprotein and intermediatedensity lipoprotein, or in very low density lipoprotein in a test samplein the above described first and second steps. However, the method maybe carried out in two steps (two-step method, two-reagent system) or inthree or more steps (multiple-step method, multiple-reagent system).

The measurement reaction may be initiated either by addition of asubstrate or substances essential to the measurement reaction or byaddition of a test sample.

The measurement operations may be performed in any set temperaturerange, such as at 30° C. or 37° C., where the reactions involved in themeasurement progress and the ingredients involved in the reactions, suchas enzymes, are not deactivated or degenerated by heat.

In the measurement method of this invention, the generated hydrogenperoxide or reduced coenzyme may be measured either by a reaction ratemethod or by an end-point method.

When the generated hydrogen peroxide or reduced coenzyme is measured byits absorbance or the like, a wavelength at which the measurement ismade is appropriately selected from the ultraviolet, visible or infraredregion depending on the type of substance to be measured.

The absorbance or the like may be measured at a single wavelength or attwo wavelengths.

In the measurement method of this invention, measurement may be madeeither by hand or by using an instrument such as automatic analyzer.

Examples of such instruments include automatic analyzers for laboratorytests.

Examples of automatic analyzers for laboratory tests include: flow-typeautomatic analyzers such as continuous flow-type and flow injection-typeanalyzers; discrete-type automatic analyzers such as closed batch-type,open batch-type, pack-type and centrifuge-type analyzers; and drychemistry-type automatic analyzers such as film-type and strip-typeanalyzers.

One example of measurement operations performed with an instrument willbe shown below:

1′. introduce a measurement reagent used in the first step of themeasurement method of this invention (a first reagent of this invention)and a measurement reagent used in the second step of the measurementmethod of this invention (a second reagent of this invention) intorespective vessels adapted to the instrument used;

2′. place the vessels containing the respective reagents at givenpositions in the instrument;

3′. introduce a sample to be tested into a vessel adapted to theinstrument and place it at a given position in the instrument;

4′. input and set measurement conditions (measurement parameters) forreagents used and a sample to be tested, when the instrument is ananalyzer for laboratory tests;

5′. initiate the measurement;

generally, introduce the test sample and the first reagent each into areaction cell (reaction cuvette) with a pipet (probe), tube or the like,mix, and expose them to each other to form a reaction system of thefirst step, and allow the reaction of the first step to progress whilekeeping the temperature constant,

6′. measure the absorbance at a specified wavelength for the reactionsolution of the test sample and the first reagent (the reaction systemof the first step) in the reaction cell (reaction cuvette), after acertain time has elapsed (after completion of the first step reactions);

7′. introduce the second reagent into the reaction solution in thereaction cell (reaction cuvette) with a pipet (probe), a tube or thelike, mix and expose them to each other to form a reaction system of thesecond step, and allow the second step reaction to progress whilekeeping the temperature constant;

8′. measure the absorbance at a specified wavelength for the reactionsolution of the test sample, the first reagent and the second reagent(the reaction system of the second step) in the reaction cell (reactioncuvette), after a certain time has elapsed (after completion of thesecond step reactions);

9′. perform the above operations 5′ to 8′ with a reaction systemcontaining purified water instead of the above sample and measure theabsorbance of the reagent blank;

10′. subtract the difference between the absorbance value obtained bythe operation 6′ and the absorbance value of the reagent blank from thedifference between the absorbance value obtained by the operation 8′ andthe absorbance value of the reagent blank to obtain an absorbancedifference; and

11′. compare the absorbance difference obtained by the operation 10′with that of a triglyceride sample of known concentration (standardsolution), calibration curve, and calculate the concentration oftriglycerides in very low density lipoprotein and intermediate densitylipoprotein, or in very low density lipoprotein in the test sample.

II. Reagents for Measurement

II-1: General Introduction of Reagents for Measurement

The reagent for selective measurement of triglycerides contained in verylow density lipoprotein and intermediate density lipoprotein, or in verylow density lipoprotein in a test sample in accordance with thisinvention is composed of a first reagent and a second reagent describedbelow.

First Reagent:

A reagent that contains a first selective reaction promoter, which is anether compound or ester compound of a polyoxyalkylene capable ofreacting lipoprotein lipase selectively with triglycerides contained inlow density lipoprotein and high density lipoprotein; lipoproteinlipase; enzymes which catalyze a series of reactions leading to thegeneration of hydrogen peroxide or a reduced coenzyme from glycerol; andan enzyme which catalyzes the reaction leading to the conversion ofhydrogen peroxide or a reduced coenzyme into another substance, and ifnecessary, further contains a substance involved in the reaction leadingto the derivation of some signal from hydrogen peroxide or a reducedcoenzyme.

Second Reagent:

A reagent that contains a second selective reaction promoter, which iscapable of reacting lipoprotein lipase selectively with triglyceridescontained in very low density lipoprotein, intermediate densitylipoprotein, low density lipoprotein and high density lipoprotein, andif necessary, further contains a substance involved in the reactionleading to the derivation of some signal from hydrogen peroxide or areduced coenzyme.

The reagent for measurement in accordance with this invention iscomposed of the above described first and second reagents; however, itmay be combined with other reagents and/or a standard substance(calibrated substance).

In the reagent in accordance with this invention, the ingredients of thefirst reagent may be divided into two or more groups and used as two ormore different reagents (in this case, the first reagent is composed oftwo or more reagents).

Similarly, the ingredients of the second reagent may be divided into twoor more groups and used as two or more different reagents (in this case,the second reagent is composed of two or more reagents).

The reagent having such a composition makes it possible to eliminatetriglycerides contained in high density lipoprotein and low densitylipoprotein in a test sample and inhibit triglycerides contained inchylomicron in the same sample from being involved in the measurementreactions at the time of measuring the test sample, wherebytriglycerides contained in very low density lipoprotein and intermediatedensity lipoprotein or in very low density lipoprotein alone aredegraded to hydrogen peroxide or a reduced coenzyme and can beselectively measured.

II-2: First Reagent

The first reagent constituting the measurement reagent in accordancewith this invention is used for performing the first step of themeasurement method of this invention (the details of the first step havealready been described in “I-2: First step”).

II-3: Second Reagent

The second reagent constituting the measurement reagent in accordancewith this invention is used for performing the second step of themeasurement method of this invention (the details of the second stephave already been described in “I-3: Second step”).

II-4: Selective Reaction Promoter

II-4-1: First Selective Reaction Promoter

The first selective reaction promoter which is contained in the firstreagent constituting the measurement reagent in accordance with thisinvention is as described in “I-4-1: First selective reaction promoter”.

II-4-2: Second Selective Reaction Promoter

The second selective reaction promoter which is contained in the secondreagent constituting the measurement reagent in accordance with thisinvention is as described in “I-4-2: Second selective reactionpromoter”.

II-4-3: Average Mole Number of Added Polyoxyalkylene in SelectiveReaction Promoters

The average mole number of the added polyoxyalkylene in the firstselective reaction promoter contained in the first reagent whichconstitutes the measurement reagent in accordance with this inventionand in the second selective reaction promoter contained in the secondreagent which constitutes the measurement reagent in accordance withthis invention are as described in “I-4-3: Average mole number of addedpolyoxyalkylene in selective reaction promoters”

II-5: Lipoprotein Lipase

The lipoprotein lipase which is contained in the first reagentconstituting the measurement reagent of this invention is as describedin “I-5-2: Reaction catalyzed by lipoprotein lipase”.

Lipoprotein lipase may be contained not only in the first reagent butalso in both of the first reagent and the second reagent.

II-6: Enzyme Catalyzing a Series of Reactions Leading to the Generationof Hydrogen Peroxide or a Reduced Coenzyme from Glycerol

The enzyme which is contained in the first reagent constituting themeasurement reagent of this invention and catalyzes a series ofreactions leading to the generation of hydrogen peroxide or a reducedcoenzyme from glycerol may be any enzyme as long as it catalyzes thereactions leading to the generation of hydrogen peroxide or a reducedcoenzyme from glycerol generated by the hydrolysis by lipoproteinlipase. The enzyme may be composed of a single kind of enzyme thatcatalyzes the series of reactions or composed of more than one kind ofenzyme that is involved in the series of reactions.

Examples of enzymes catalyzing the series of reactions leading to thegeneration of hydrogen peroxide or a reduced coenzyme from glycerolinclude glycerol kinase, glycerol-3-phosphate oxidase andglycerol-3-phosphate dehydrogenase.

The details of the series of reactions leading to the generation ofhydrogen peroxide or a reduced coenzyme from glycerol and those of theenzymes catalyzing the reactions are as described in “I-5-3: A series ofreactions leading to the generation of hydrogen peroxide or reducedcoenzyme from glycerol”.

The enzyme catalyzing a series of reactions leading to the generation ofhydrogen peroxide or a reduced coenzyme from glycerol may be containednot only in the first reagent but also in both of the first reagent andthe second reagent.

The catalytic reaction by glycerol kinase requires adenosinetriphosphate (ATP) and magnesium ion. Accordingly, when using glycerolkinase, adenosine triphosphate and magnesium ion are present (orcontained) in the first reagent.

The details of adenosine triphosphate (ATP) and magnesium ion are asdescribed in “I-5-3: A series of reactions leading to the generation ofhydrogen peroxide or reduced coenzyme from glycerol”

The adenosine triphosphate (ATP) or the magnesium ion may be containednot only in the first reagent but also in both of the first reagent andthe second reagent.

The catalytic reaction by glycerol-3-phosphate dehydrogenase requires anoxidized coenzyme such as nicotinamide adenine dinucleotide (oxidizedform) [NAD⁺] or nicotinamide adenine dinucleotide phosphate (oxidizedform) [NADP⁺ (oxidized)]. Accordingly, when using glycerol-3-phosphatedehydrogenase, the oxidized coenzyme is added to the first reagent (orthe first and second reagents).

II-7: Enzymes Catalyzing the Reaction Leading to the Conversion ofHydrogen Peroxide or a Reduced Coenzyme into another Substance

The enzyme which is contained in the first reagent constituting themeasurement reagent of this invention and catalyzes a reaction leadingto the conversion of hydrogen peroxide or a reduced coenzyme intoanother substance may be any enzyme as long as it can catalyze thereaction leading to the conversion of the generated hydrogen peroxide ora reduced coenzyme into another substance. The enzyme may be composed ofa single kind of enzyme that catalyzes the reaction or composed of morethan one kind of enzyme that is involved in the reaction.

Examples of enzymes which catalyze the reaction leading to theconversion of hydrogen peroxide into another substance include catalaseand peroxidase.

Examples of enzymes which catalyze the reaction leading to theconversion of a reduced coenzyme into another substance includedehydrogenase, which catalyzes the reaction leading to the conversion ofthe reduced coenzyme into an oxidized coenzyme using the reducedcoenzyme as a coenzyme. More specifically, they include lactatedehydrogenase, malate dehydrogenase and isocitrate dehydrogenase.

When adding catalase to the first reagent and using it as an enzyme thatcatalyzes the reaction leading to the conversion of hydrogen peroxideinto another substance, a substance that inhibits the activity ofcatalase, such as sodium azide, should be added to the second reagent sothat the hydrogen peroxide generated in the second step is noteliminated (degraded) by the catalase.

When adding peroxidase to the first reagent and using it as an enzymethat catalyzes the reaction leading to the conversion of hydrogenperoxide into another substance, an oxidizable substance also needs beadded to the first reagent.

This allows hydrogen peroxide and the oxidizable substance to beconverted into oxidized substances through the catalytic reaction byperoxidase.

Examples of oxidizable substances include 4-aminoantipyrine, phenol orderivatives thereof, or aniline derivatives.

If both 4-aminoantipyrine and phenol or derivative thereof or both4-aminoantipyrine and an aniline derivative exist together in the firstreagent, color is developed in the first step by the generated hydrogenperoxide and peroxidase; therefore, either 4-aminoantipyrine or phenolor derivative thereof alone, or either 4-aminoantipyrine or an anilinederivative alone must be present (contained) in the first reagent.

The details of the reaction leading to the conversion of hydrogenperoxide or a reduced coenzyme into another substance and those of theenzyme catalyzing the reaction are as described in “I-6: Reactionleading to the conversion of hydrogen peroxide or reduced coenzyme intoanother substance”.

II-8: Substances Involved in the Reaction Leading to the Derivation ofsome Signal from Hydrogen Peroxide or Reduced Coenzyme

Substances involved in the reaction leading to the derivation of somesignal from hydrogen peroxide or reduced coenzyme may be added,depending on the situation, to the first reagent, the second reagent, orboth the first and second reagents which constitute the measurementreagent of this invention.

In measurement of the hydrogen peroxide or reduced coenzyme which isgenerated by mixing and catalytically reacting a test sample with thefirst reagent (the first step) and further mixing and catalyticallyreacting the test sample with the second reagent (the second step),except in case the generated hydrogen peroxide itself or the generatedreduced coenzyme itself is measured, a substance involved in thereaction leading to the derivation of some signal from the hydrogenperoxide or reduced coenzyme needs to be contained in the first reagent,the second reagent, or both the first and second reagents.

The substance involved in the reaction leading to the derivation of somesignal from the hydrogen peroxide or reduced coenzyme may be anysubstance as long as it is involved in the reaction leading to thederivation of some signal from the generated hydrogen peroxide orreduced coenzyme. The substance may be composed of a single substanceinvolved in the above described reaction or composed of more than onesubstance involved in the above described reaction.

Examples of substances involved in the reaction leading to thederivation of some signal from hydrogen peroxide or a reduced coenzymeinclude peroxidase, chromogens in a Trinder reaction system(4-aminoantipyrine, phenol or derivatives thereof, and anilinederivatives), diaphorase, 1-methoxy-phenazine methosulfate, andtetrazolium salts.

All the substances involved in the reaction leading to the derivation ofsome signal from hydrogen peroxide or a reduced coenzyme may be added toone reagent, either the first reagent or the second reagent; however,from the viewpoint of the substance stability, preferable they aredistributed between the two reagents.

For example, the following two substances: 4-aminoantipyrine and phenolor derivative thereof, or 4-aminoantipyrine and an aniline derivativeare separated from each other and distributed between the first andsecond reagents.

If the following three substances: peroxidase, 4-aminoantipyrine andphenol or derivative thereof, or peroxidase, 4-aminoantipyrine and ananiline derivative are contained together in the first reagent, color isdeveloped by the generated hydrogen peroxide in the first step;therefore, preferably the above described three substances aredistributed between the first and second reagents.

The details of the substances involved in the reaction leading to thederivation of some signal from hydrogen peroxide or a reduced coenzymeis as described in “I-7: Measurement of hydrogen peroxide or reducedcoenzyme”.

II-9: Reaction Assistant

In the measurement reagent of this invention, the first and/or thesecond reagent may contain a reaction assistant.

The details of the reaction assistant are as described in “I-8: Reactionassistant”.

II-10: Other substances

The first and/or the second reagent constituting the measurement reagentof this invention may contain, depending on the situation, additionalsubstances such as buffers; enzymes other than the above described ones;substrates for the other enzymes; coenzymes other than the abovedescribed ones; ions or salts of alkaline metals, alkaline earth metalsor the like; chelators; proteins such as albumin; sodium azide,antibiotics or preservatives such as a synthetic antibacterial agent;stabilizers such as saccharides or polymer compounds; activators;substances involved in the elimination or inhibition of measurementinterfering substances contained in a test sample, such as ascorbateoxidase; excipients; and other reagent ingredients.

Preferably the pH values of the first and the second reagentsconstituting the measurement reagent of this invention are in the rangeof 5 to 10 and particularly preferably in the range of 5.5 to 9.0.

Accordingly, it is preferable the first and the second reagents eachcontain a buffer that allows their pH values to fall in the abovedescribed range.

Examples of such buffers are as described in “I-9: Other substances”.

II-11: Test Sample

The details of the samples to be tested with the measurement reagent ofthis invention are as described in “I-10: Test samples”.

II-12: Measurement operations

The details of the operations for selective measurement, using themeasurement reagent of this invention, of triglycerides contained invery low density lipoprotein and intermediate density lipoprotein or invery low density lipoprotein in the test sample are as described in“I-11: Measurement operations”.

This specification includes part or all of the contents as disclosed inthe specification of Japanese Patent Application No. 2002-168738, whichis a priority document of the present application.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail giving severalexamples. The examples are not intended to limit the scope of thisinvention.

EXAMPLE 1

Measurement of triglycerides in purified lipoprotein fractions using themeasurement method and reagent of this invention

Triglycerides in purified lipoprotein fractions were measured using themeasurement method and reagent of this invention while varyingsubstances used as the first selective reaction promoter and the secondselective reaction promoter.

1. Preparation of Measurement Reagent of this Invention

1-1: Preparation of First Reagent (Reagent A)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent A ofpH 6.0 (20° C.). Reagent ingredient Concentation2-Morpholinoethanesulfonic acid [MES] 50 mMN-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline 1.5 mM sodium salt(chromogen) Glycerol kinase 150 units/l Glycerol-3-phosphate oxidase3,000 units/l Sodium adenosine triphosphate 0.5 mM Magnesium chloridehexahydrate 1 mM Catalase 100,000 units/l Lipoprotein lipase [LP-BP(Asahi Kasei Corporation)] 300,000 units/l First selective reactionpromoter [name of each 0.1% (w/v) substance is shown in Table 1]

TABLE 1 Supplier Chemical structure First selective reaction promoterNP-10 Nikko Chemicals Co., Ltd. Polyoxyethylene nonylphenyl ether (10)Emulgen 911 Kao Corporation Polyoxyethylene nonylphenyl ether (11)NP-11.2 Nikko Chemicals Co., Ltd. Polyoxyethylene nonylphenyl ether(11.2) NP-11.4 Nikko Chemicals Co., Ltd. Polyoxyethylene nonylphenylether (11.4) NP-11.5 Nikko Chemicals Co., Ltd. Polyoxyethylenenonylphenyl ether (11.5) NP-11.6 Nikko Chemicals Co., Ltd.Polyoxyethylene nonylphenyl ether (11.6) NP-13 Nikko Chemicals Co., Ltd.Polyoxyethylene nonylphenyl ether (13) Second selective reactionpromoter NP-10 Nikko Chemicals Co., Ltd. Polyoxyethylene nonylphenylether (10) NP-11.2 Nikko Chemicals Co., Ltd. Polyoxyethylene nonylphenylether (11.2) NP-11.4 Nikko Chemicals Co., Ltd. Polyoxyethylenenonylphenyl ether (11.4) NP-11.6 Nikko Chemicals Co., Ltd.Polyoxyethylene nonylphenyl ether (11.6) NP-13 Nikko Chemicals Co., Ltd.Polyoxyethylene nonylphenyl ether (13)*The values in parentheses indicate the average mole number of the addedpolyoxyethylene.

1-2: Preparation of Second Reagent (Reagent B)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent B ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LPL (Asahi KaseiCorporation)] 500,000 units/l Sodium azide 0.1% (w/v) Second selectivereaction promoter [name of each 0.5% (w/v) substance is shown in Table1]

2. Preparation of Reagent for Total Triglyceride Measurement (Control)

2-1: Preparation of First Reagent (Reagent C)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent C ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mMN-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline 1.5 mM sodium salt(chromogen) Glycerol kinase 150 units/l Glycerol-3-phosphate oxidase3,000 units/l Sodium adenosine triphosphate 0.5 mM Magnesium chloridehexahydrate 1 mM Catalase 100,000 units/l Adekanol B-795 (Asahi DenkaKogyo K.K.) 0.5% (w/v)

2-2: Preparation of Second Reagent (Reagent D)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent D ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LPL (Asahi KaseiCorporation)] 120,000 units/l Sodium azide 0.1% (w/v) Adekanol B-795(Asahi Denka Kogyo K.K.) 0.5% (w/v)

3. Preparation of Purified Lipoprotein Fractions

Blood was collected in a tube containing an anticoagulant and separatedby density-gradient centrifugation into 5 types of lipoprotein fractionshaving different densities: chylomicron, very low density lipoprotein,intermediate density lipoprotein, low density lipoprotein and highdensity lipoprotein. These five types of fractions were used as testsamples in the measurement of triglycerides.

4. Measurement of Triglycerides in Lipoprotein Fractions

The level of triglycerides in each lipoprotein fraction prepared insection 3 was measured using a Hitachi automatic analyzer 7170S(Hitachi, Ltd.) through the following procedure:

1′. Dispense 2.5 μl of each lipoprotein fraction prepared in section 3,as a test sample, in a reaction cell (reaction cuvette).

Then, dispense 200 μl of the first reagent of this invention (Reagent A)prepared in section 1-1 in the reaction cell (reaction cuvette) and mixit with the test sample.

Warm up the reaction cell (reaction cuvette) at 37° C. for the reactionof the first step.

2′. Four minutes 30 seconds (16th point) after the addition of the firstreagent (Reagent A), measure the absorbance (main wavelength 600 nm, subwavelength 700 nm) of the reaction solution (the reaction solution ofthe test sample and the first reagent) in the reaction cell (reactioncuvette) to give a sample blank.

3′. After the measurement of the absorbance, dispense 100 μl of thesecond reagent of this invention (Reagent B) prepared in section 1-2 inthe reaction solution in the reaction cell (reaction cuvette) and mix itwith the reaction solution in the cell (cuvette).

Then, warm up the reaction cell (reaction cuvette) at 37° C. for thereaction of the second step.

4′. Nine minutes 47 seconds (34th point) after the addition of the firstreagent (Reagent A), measure the absorbance (main wavelength 600 nm, subwavelength 700 nm) of the reaction solution (the reaction solution ofthe test sample, the first reagent and the second reagent) in thereaction cell (reaction cuvette) to give a measured value of the testsample.

5′. Repeat the operating procedures 1′ to 4′ for the reaction solutionin which the test sample has been replaced by purified water to give ameasured absorbance of a reagent blank.

6′. Calculate the absorbance difference of the test sample bysubtracting the difference between the absorbance obtained in procedure2′ (sample blank) and the absorbance of the reagent blank from thedifference between the absorbance obtained in procedure 4′ (measuredvalue) and the absorbance of the reagent blank.

7′. Perform the same operating procedures as 1′ to 6′ using: an aqueoussolution of triolein (trioleoylglycerol) (concentration: 250 mg/dl),which is a kind of triglyceride, as a test sample; the first reagent ofthe total triglyceride measurement reagent (Reagent C) prepared in 2-1;and the second reagent of the total triglyceride measurement reagent(Reagent D) prepared in 2-2 to calculate the absorbance difference ofthe aqueous solution of triolein [calibration curve].

8′. Compare the absorbance difference of the test sample calculated inthe operating procedure 6′ with that of the aqueous solution of triolein(250 mg/dl) calculated in procedure 7′ [calibration curve] and calculatethe concentration of triglycerides contained in the test sample (thelipoprotein fraction).

The combinations of the first selective reaction promoter contained inthe first reagent (Reagent A) and the second selective reaction promotercontained in the second reagent (Reagent B) used in the measurement oftriglycerides are shown in Table 2.

The level of triglycerides contained in each lipoprotein fractionprepared in 3 was measured using the first reagent of the totaltriglyceride measurement reagent prepared in 2-1 (Reagent C) and thesecond reagent of the total triglyceride measurement reagent prepared in2-2 (Reagent D) in the same manner as described above.

The effect of the first and the second selective reaction promotersadded into the measurement reagent of this invention was confirmed bythe values obtained by dividing the level (measured value) oftriglycerides contained in each test sample (lipoprotein fraction) whenusing the measurement reagent of this invention (Reagent A and ReagentB) by the level (measured value) of triglycerides contained in each testsample (lipoprotein fraction) when using the total triglyceridemeasurement reagent (Reagent C and Reagent D).

The values are shown in Table 2. TABLE 2 Second First selectiveselective reaction reaction promoter promoter contained in firstcontained in reagent second reagent Test sample (Lipoprotein fraction)(Reagent A) (Reagent B) CM VLDL IDL LDL HDL NP-10 NP-10 0.05 0.01 0.030.04 0.01 Emulgen 911 NP-10 0.08 0.69 0.63 0.13 0.03 NP-11.2 NP-10 0.070.48 0.44 0.07 0.01 NP-11.4 NP-10 0.07 0.62 0.56 0.09 0.03 NP-11.5 NP-100.08 0.69 0.64 0.18 0.03 NP-11.6 NP-10 0.08 0.69 0.64 0.17 0.02 NP-13NP-10 0.08 0.72 0.65 0.50 0.04 Emulgen 911 NP-10 0.07 0.61 0.49 0.070.01 Emulgen 911 NP-11.2 0.03 0.29 0.35 0.06 0.00 Emulgen 911 NP-11.40.03 0.26 0.32 0.05 0.02 Emulgen 911 NP-11.6 0.03 0.23 0.28 0.05 0.02Emulgen 911 NP-13 0.03 0.16 0.17 0.04 0.02 Reagents for measuring total1.00 1.00 1.00 1.00 1.00 triglyceride (Reagent C and Reagent D)[Control]CM: ChylomicronVLDL: Very low density lipoproteinIDL: Intermediate density lipoproteinLDL: Low density lipoproteinHDL: High density lipoprotein

5. SUMMARY

5-1: When the Second Selective Reaction Promoter is NP-10

The measured results will be described below which were obtained usingNP-10 (polyoxyethylene nonylphenyl ether in which the average molenumber of the added polyoxyethylene is 10) as the second selectivereaction promoter contained in the second reagent (Reagent B) of thisinvention while varying the average mole number of the addedpolyoxyethylene in the first selective reaction promoter(polyoxyethylene nonylphenyl ether) contained in the first reagent ofthis invention (Reagent A).

1′. The results show that when the average mole number of the addedpolyoxyethylene in the first selective reaction promoter was 10 [NP-10],triglycerides contained in each lipoprotein fraction could not bemeasured in any one of the lipoprotein fractions: chylomicron fraction,very low density lipoprotein fraction, intermediate density lipoproteinfraction, low density lipoprotein fraction and high density lipoproteinfraction.

2′. The results also show that when the average mole number of the addedpolyoxyethylene in the first selective reaction promoter was 11 [Emulgen911], 11.2 [NP-11.2], 11.4 [NP-11.4], 11.5 [NP-11.5] or 11.6 [NP-11.6],triglycerides could be measured in the lipoprotein fractions such asvery low density lipoprotein fraction and intermediate densitylipoprotein fraction, while they was hardly measured or only a verysmall amount of them was measured in the lipoprotein fractions such aschylomicron fraction, low density lipoprotein fraction and high densitylipoprotein fraction.

This confirms that where the average mole number of the addedpolyoxyethylene is rounded to the nearest whole number, when the averagemole number (n) of the added polyoxyethylene in the second selectivereaction promoter is 10 and the average mole number (m) of the addedpolyoxyethylene in the first selective reaction promoter is 11 to 12(when the m/n ratio is 1.1 to 1.2), triglycerides contained in very lowdensity lipoprotein and intermediate density lipoprotein can beselectively measured, compared with those contained in chylomicron, lowdensity lipoprotein and high density lipoprotein.

3′. The results show that when the average mole number of the addedpolyoxyethylene in the first selective reaction promoter was 13 [NP-13],not only triglycerides contained in the very low density lipoproteinfraction and the intermediate density lipoprotein fraction, buttriglycerides contained in the low density lipoprotein fraction weremeasured.

This confirms that with NP-13, triglycerides contained in very lowdensity lipoprotein and intermediate density lipoprotein or in very lowdensity lipoprotein cannot be selectively measured.

5-2: When the First Selective Reaction Promoter is Emulgen 911

The measured results will be described below which were obtained usingEmulgen 911 (polyoxyethylene nonylphenyl ether in which the average molenumber of the added polyoxyethylene is 11) as the first selectivereaction promoter contained in the first reagent (Reagent A) of thisinvention while varying the average mole number of the addedpolyoxyethylene in the second selective reaction promoter(polyoxyethylene nonylphenyl ether) contained in the second reagent ofthis invention (Reagent B).

1′. The results show that when the average mole number of the addedpolyoxyethylene in the second selective reaction promoter was 10[NP-10], triglycerides contained in the very low density lipoproteinfraction and the intermediate density lipoprotein fraction could bemeasured, while triglycerides contained in the chylomicron fraction, thelow density lipoprotein fraction and the high density lipoproteinfraction could hardly be measured.

The experimental results also confirms that when the average mole number(m) of the added polyoxyethylene in the first selective reactionpromoter is 11 and the average mole number (n) of the addedpolyoxyethylene in the second selective reaction promoter is 10 (whenthe m/n ratio is 1.1), triglycerides contained in very low densitylipoprotein and intermediate density lipoprotein can be selectivelymeasured, compared with those contained in chylomicron, low densitylipoprotein and high density lipoprotein.

2′. The results also show that when the average mole number of the addedpolyoxyethylene in the second selective reaction promoter was 11.2[NP-11.2], 11.4 [NP-11.4], 11.6 [NP-11.6] or 13 [NP-13], triglyceridescould be measured in the lipoprotein fractions such as very low densitylipoprotein fraction and intermediate density lipoprotein fraction;however, the percentage was low.

This confirms that where the average mole number of the addedpolyoxyethylene is rounded to the nearest whole number, the measurementreagents in which the average mole number (m) of the addedpolyoxyethylene in the first selective reaction promoter is 11 and theaverage mole number (n) of the added polyoxyethylene in the secondselective reaction promoter is 11 to 13 (when the m/n ratio is 0.85 to1.0) are not suitably used for the selective measurement oftriglycerides contained in very low density lipoprotein and intermediatedensity lipoprotein or in very low density lipoprotein.

EXAMPLE 2

Confirmation of Effect of Reaction Assistants

The effect of the presence (addition) of a reaction assistant on theselective-reaction-promotion activity of the first selective reactionpromoter and/or the second selective reaction promoter was confirmed.

1. Preparation of Measurement Reagent of this Invention

1-1: Preparation of First Reagent (Reagent E)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent E ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mMN-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline 1.5 mM sodium salt(chromogen) Glycerol kinase 150 units/l Glycerol-3-phosphate oxidase3,000 units/l Sodium adenosine triphosphate 0.5 mM Magnesium chloridehexahydrate 1 mM Catalase 100,000 units/l Lipoprotein lipase [LP-BP(Asahi Kasei Corporation)] 100,000 units/l Emulgen 911 (Kao Corporation)0.1% (w/v)

Reaction assistants (the substance name and the concentration of eachassistant is shown in Table 3) TABLE 3 Reaction assistant Chemicalstructure Concentration β-CD β-cyclodextrin 0.01% 0.05% 0.10% γ-CDγ-cyclodextrin 0.01% 0.05% 0.10% Hydroxypropyl-γ-CDHydroxypropyl-γ-cyclodextrin 0.01% (Nihon Shokuhin Kako Co., Ltd.) 0.05%0.10% Sodium α-CD sulfonate Sodium α-cyclodextrin sulfonate 0.01% 0.05%0.10% ISOELEAT P The total amount of cyclodextrin 0.01% (Bio ResearchCorporation of constitutes 80% or more of ISOELEAT P 0.05% Yokohama) and50% or more of the total cyclodextrin 0.10% is maltosyl cyclodextrin.Maltosyl-β-CD Maltosyl-β-cyclodextrin 0.01% 0.05% 0.10% Water-solubleβ-cyclodextrin Water-soluble β-cyclodextrin polymer 0.01% polymer(epichlorohydrin crosslinked) 0.05% (Nihon Shokuhin Kako Co., Ltd.)0.10% Water-soluble γ-cyclodextrin Water-soluble γ-cyclodextrin polymer0.01% polymer (epichlorohydrin crosslinked) 0.05% (Nihon Shokuhin KakoCo., Ltd.) 0.10%None (no reaction assistant added)

1-2: Preparation of Second Reagent (Reagent F)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent F ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LPL (Asahi KaseiCorporation)] 500,000 units/l Sodium azide 0.1% (w/v) NP-10 (NikkoChemicals Co., Ltd.) 0.5% (w/v)

2. Preparation of Reagent for Total Triglyceride Measurement (Control)

2-1: Preparation of First Reagent (Reagent C)

The first reagent (Reagent C) prepared in Example 1, section 2-1 wasused.

2-2: Preparation of Second Reagent (Reagent D)

The second reagent (Reagent D) prepared in Example 1, section 2-2 wasused.

3. Purified lipoprotein fractions

The five types of lipoprotein fractions: chylomicron, very low densitylipoprotein, intermediate density lipoprotein, low density lipoproteinand high density lipoprotein fractions prepared in Example 1, section 3were used as test samples in the measurement of triglycerides.

4. Measurement of Triglycerides in Lipoprotein Fractions

The level of triglycerides in each lipoprotein fraction prepared insection 3 was measured through the same operating procedures asprocedures 1′ to 8′ described in Example 1, section 4, except that thefirst reagent of this invention (Reagent A) was replaced by the firstreagent of this invention (Reagent E) prepared in 1-1 and the secondreagent of this invention (Reagent B) was replaced by the second reagentof this invention (Reagent F) prepared in 1-2.

Measurement of triglycerides was made for the cases using the abovedescribed kinds of first reagent (Reagent E) (for the cases using theabove described reaction assistants and concentrations).

The level of triglycerides contained in the lipoprotein fractionsprepared in section 3 was also measured in the same manner as above,using the first reagent of the total triglyceride measurement reagent(Reagent C) prepared in section 2-1 and the second reagent of the totaltriglyceride measurement reagent (Reagent D) prepared in section 2-2.

The level (measured value) of triglycerides in each test sample(lipoprotein fraction) measured using the measurement reagent of thisinvention (Reagent E and Reagent F) was divided by the level (measuredvalue) of triglycerides in the same test sample (lipoprotein fraction)measured using the total triglyceride measurement reagent (Reagent C andReagent D).

The values are shown in Table 4. TABLE 4 Concen- Test sample(Lipoprotein fraction) Reaction assistant tration CM VLDL IDL LDL HDLβ-CD 0.01% 0.05 0.72 0.61 0.23 0.03 0.05% 0.05 0.67 0.54 0.15 −0.04 0.1% 0.05 0.59 0.49 0.18 0.02 γ-CD 0.01% 0.05 0.72 0.60 0.33 0.01 0.05%0.05 0.70 0.56 0.19 0.02  0.1% 0.05 0.63 0.52 0.18 0.03Hydroxypropyl-γ-CD 0.01% 0.05 0.72 0.62 0.22 0.04 (Nihon Shokuhin Kako0.05% 0.05 0.71 0.58 0.20 0.04 Co., Ltd.) 0.1% 0.05 0.67 0.53 0.16 0.03Sodium α-CD sulfonate 0.01% 0.05 0.73 0.61 0.21 0.01 0.05% 0.05 0.720.62 0.21 0.04  0.1% 0.05 0.72 0.61 0.21 0.02 ISOELEAT P 0.01% 0.05 0.720.61 0.25 0.04 (Bio Research 0.05% 0.05 0.72 0.59 0.23 0.05 Corporationof  0.1% 0.05 0.71 0.59 0.19 0.06 Yokohama) Maltosyl-β-CD 0.01% 0.050.72 0.61 0.25 0.04 0.05% 0.05 0.69 0.57 0.19 0.04  0.1% 0.05 0.58 0.500.18 0.05 Water-soluble 0.01% 0.05 0.74 0.63 0.25 0.06 β-cyclodextrinpolymer 0.05% 0.05 0.69 0.57 0.18 0.04 (Nihon Shokuhin Kako  0.1% 0.050.60 0.52 0.17 0.04 Co., Ltd.) Water-soluble 0.01% 0.05 0.75 0.61 0.260.05 γ-cyclodextrin polymer 0.05% 0.05 0.72 0.60 0.22 0.05 (NihonShokuhin Kako  0.1% 0.05 0.69 0.57 0.19 0.03 Co., Ltd.) None (noreaction assistant added) 0.05 0.72 0.61 0.28 0.04 Reagents formeasuring total 1.00 1.00 1.00 1.00 1.00 triglyceride (Reagent C andReagent D) [Control]CM: ChylomicronVLDL: Very low density lipoproteinIDL: Intermediate density lipoproteinLDL: Low density lipoproteinHDL: High density lipoprotein

5. SUMMARY

1′. The results indicate that the addition of β-CD as a reactantassistant into the first reagent of this invention (Reagent E) madepossible the decrease in degree to which triglycerides contained in lowdensity lipoprotein were measured.

The results also indicate that although the increase in theconcentration of β-CD decreased the degree to which triglyceridescontained in very low density lipoprotein and intermediate densitylipoprotein were measured, the rate of the decrease was less than thatin the case of triglycerides in low density lipoprotein measured.

The use of hydroxypropyl-γ-CD, maltosyl-β-CD, water-solubleβ-cyclodextrin polymer or water-soluble γ-cyclodextrin polymer as areaction assistant produced the same effects.

2′. The results indicate that the addition of γ-CD as a reactionassistant into the first reagent of this invention (Reagent E) madepossible the decrease in degree to which triglycerides contained in lowdensity lipoprotein were measured.

Although the increase in the concentration of γ-CD decreased the degreeto which triglycerides contained in very low density lipoprotein andintermediate density lipoprotein were measured, the rate of the decreasewas less than that in the case of triglycerides in low densitylipoprotein measured.

3′. The results indicate that the addition of sodium α-CD sulfonate as areaction assistant into the first reagent of this invention (Reagent E)made possible the decrease in degree to which triglycerides contained inlow density lipoprotein were measured.

4′. The results also indicate that the addition of ISOELEAT P as areaction assistant into the first reagent of this invention (Reagent E)made possible the decrease in degree to which triglycerides contained inlow density lipoprotein were measured.

The increase in the concentration of ISOELEAT P added also made possiblethe decrease in degree to which triglycerides contained in low densitylipoprotein were measured.

The results described so far confirm that the presence (addition) ofcyclodextrin or derivative thereof as a reaction assistant makes itpossible to enhance the selective-reaction-promotion activity of thefirst selective reaction promoter and/or the second selective reactionpromoter.

EXAMPLE 3

Confirmation of Effects of Lipoprotein Lipase's Character

The effects of characters of of the lipoprotein lipase contained in thefirst reagent (present in the first step) and the lipoprotein lipasecontained in the second reagent (present in the second step) on themeasurement results were confirmed.

1. Preparation of Measurement Reagent of this Invention

1-1: Preparation of First Reagent (Reagent G)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent G ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mMN-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline 1.5 mM sodium salt(chromogen) Glycerol kinase 150 units/l Glycerol-3-phosphate oxidase3,000 units/l Sodium adenosine triphosphate 0.5 mM Magnesium chloridehexahydrate 1 mM Catalase 100,000 units/l Emulgen 911 (Kao Corporation)0.1% (w/v)

Lipoprotein lipase [the trade name and the activity level of each lipaseare shown in Table 5]

1-2: Preparation of Second Reagent (Reagent H)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent H ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-aminoantipyrine 0.75 mMPeroxidase 600 units/l Sodium azide 0.1% (w/v) NP-10 (Nikko ChemicalsCo., Ltd.) 0.5% (w/v)

Lipoprotein lipase [the trade name and the activity level of each lipaseare shown in Table 5]

2. Preparation of Reagent for Total Triglyceride Measurement (Control)

2-1: First Reagent (Reagent C)

The first reagent (Reagent C) prepared in Example 1, section 2-1 wasused.

2-2: Second Reagent (Reagent D)

The second reagent (Reagent D) prepared in Example 1, section 2-2 wasused.

3. Purified Lipoprotein Fractions

The five types of lipoprotein fractions: chylomicron, very low densitylipoprotein, intermediate density lipoprotein, low density lipoproteinand high density lipoprotein prepared in Example 1, section 3 were usedas test samples in the measurement of triglycerides.

4. Measurement of Triglycerides in Lipoprotein Fractions

The level of triglycerides in each lipoprotein fraction prepared insection 3 was measured through the same operating procedures asprocedures 1′ to 8′ described in Example 1, section 4, except that thefirst reagent of this invention (Reagent A) was replaced by the firstreagent of this invention (Reagent G) prepared in section 1-1 and thesecond reagent of this invention (Reagent B) was replaced by the secondreagent of this invention (Reagent H) prepared in section 1-2.

The combinations of the lipoprotein lipase contained in the firstreagent of this invention (Reagent G) and the lipoprotein lipasecontained in the second reagent of this invention (Reagent H) in themeasurement as above are as shown in Table 5.

The level of triglycerides in each lipoprotein fraction prepared insection 3 was also measured in the same manner as above, using the firstreagent of the total triglyceride measurement reagent (Reagent C) insection 2-1 and the second reagent of the total triglyceride measurementreagent (Reagent D) in section 2-2.

The level (measured value) of triglycerides in each test sample(lipoprotein fraction) measured using the measurement reagent of thisinvention (Reagent G and Reagent H) was divided by the level (measuredvalue) of triglycerides in the same test sample (lipoprotein fraction)measured using the total triglyceride measurement reagent (Reagent C andReagent D).

The values are shown in Table 5. TABLE 5 Lipoprotein lipase Lipoproteinlipase contained in the first contained in the second Test sample(lipoprotein fraction) reagent (Reagent G) reagent (Reagent H) CM VLDLIDL LDL HDL LP-BP [50,000 units/l] LPL [500,000 units/l] 0.02 0.67 0.550.30 0.03 LP-BP [100,000 units/l] LPL [500,000 units/l] 0.02 0.66 0.550.22 0.03 LP-BP [250,000 units/l] LPL [500,000 units/l] 0.02 0.68 0.560.18 0.03 LP-BP [500,000 units/l] LPL [500,000 units/l] 0.01 0.66 0.530.15 0.01 LPL-314 [100 units/l] LPL [500,000 units/l] 0.03 0.64 0.550.28 0.05 LPL-314 [500 units/l] LPL [500,000 units/l] 0.02 0.63 0.490.17 0.02 LPL-314 [1,000 units/l] LPL [500,000 units/l] 0.02 0.63 0.450.14 0.03 LP-BP [50,000 units/l] LPL-314 [100 units/l] 0.01 0.46 0.470.28 0.03 LP-BP [50,000 units/l] LPL-314 [500 units/l] 0.01 0.49 0.480.27 0.01 LP-BP [50,000 units/l] LPL-314 [1,000 units/l] 0.02 0.50 0.490.28 0.03 LPL-311 [100 units/l] LPL [500,000 units/l] 0.04 0.64 0.550.28 0.06 LPL-311 [500 units/l] LPL [500,000 units/l] 0.02 0.59 0.410.15 0.01 LPL-311 [1,000 units/l] LPL [500,000 units/l] 0.02 0.48 0.300.12 0.03 Total triglyceride measurementreagents(Reagent 1.00 1.00 1.001.00 1.00 C and Reagent D)[control]* Lipoprotein lipase whose activity depends on the presence of asurfactant.LP-BP (Asahi Kasei Corporation)LPL-314 (Toyobo Co., Ltd.)* Lipoprotein lipase whose activity hardly depends on the presence of asurfactant.LPL (Asahi Kasei Corporation)LPL-311 (Toyobo Co., Ltd.)CM: ChylomicronVLDL: Very low density lipoproteinIDL: Intermediate density lipoproteinLDL: Low density lipoproteinHDL: High density lipoprotein

5. SUMMARY

1′. The results indicate that when LP-BP (Asahi Kasei Corporation), alipoprotein lipase whose activity depends on the concentration of asurfactant, was contained in the first reagent (present in the firststep) and LPL (Asahi Kasei Corporation), a lipoprotein lipase whoseactivity hardly depends on the concentration of a surfactant, wascontained in the second reagent (present in the second step),triglycerides contained in the very low density lipoprotein fractionsand intermediate density lipoprotein fractions could be measured, whiletriglycerides contained in the chylomicron fractions, low densitylipoprotein fractions and high density lipoprotein fractions couldhardly be measured or be measured only in a very small amount.

The results also indicate that the degree to which triglyceridescontained in low density lipoprotein fractions were measured wasdecreased with the increase in the activity level of LP-BP contained inthe first reagent (present in the first step).

2′. The results indicate that when LPL-314 (Toyobo Co., Ltd.), alipoprotein lipase whose activity depends on the concentration of asurfactant, was contained in the first reagent (present in the firststep) and LPL (Asahi Kasei Corporation), a lipoprotein lipase whoseactivity hardly depends on the concentration of a surfactant, wascontained in the second reagent (present in the second step),triglycerides contained in the very low density lipoprotein fractionsand intermediate density lipoprotein fractions could be measured, whiletriglycerides contained in the chylomicron fractions, low densitylipoprotein fractions and high density lipoprotein fractions couldhardly be measured or be measured only in a very small amount.

The results also indicate that the degree to which triglyceridescontained in low density lipoprotein fractions were measured wasdecreased with the increase in the activity level of LPL-314 containedin the first reagent (present in the first step).

3′. The results indicate that when LP-BP (Asahi Kasei Corporation), alipoprotein lipase whose activity depends on the concentration of asurfactant, was contained in the first reagent (present in the firststep) and LPL-314 (Toyobo Co., Ltd.), also a lipoprotein lipase whoseactivity depends on the concentration of a surfactant, was contained inthe second reagent (present in the second step), triglycerides containedin the very low density lipoprotein fractions and intermediate densitylipoprotein fractions could be measured, while triglycerides containedin the chylomicron fractions, low density lipoprotein fractions and highdensity lipoprotein fractions could be measured only in a very smallamount.

However, the results show that the degree to which triglyceridescontained in very low density lipoprotein fractions and intermediatedensity lipoprotein fractions were measured was a little decreased,compared with that of 1′ and 2′.

4′. The results indicate that when LPL-311 (Toyobo Co., Ltd.), alipoprotein lipase whose activity hardly depends on the concentration ofa surfactant, was contained in the first reagent (present in the firststep) and LPL (Asahi Kasei Corporation), also a lipoprotein lipase whoseactivity hardly depends on the concentration of a surfactant, wascontained in the second reagent (present in the second step),triglycerides contained in the very low density lipoprotein fractionsand intermediate density lipoprotein fractions could be measured, whiletriglycerides contained in the chylomicron fractions, low densitylipoprotein fractions and high density lipoprotein fractions couldhardly be measured or be measured only in a very small amount.

However, the result also indicate that the degree to which triglyceridescontained in the very low density lipoprotein fractions and intermediatedensity lipoprotein fractions were measured was decreased with theincrease in the activity level of LPL-311 contained in the first reagent(present in the first step).

The results described so far confirm that lipoprotein lipase containedin the first reagent (present in the first step) is preferably of a typewhose activity depends on the concentration of a surfactant andlipoprotein lipase contained in the second reagent (present in thesecond step) is preferably of a type whose activity hardly depends onthe concentration of a surfactant.

EXPERIMENTAL EXAMPLE

Confirmation of Lipoprotein Lipase's Character That the activity dependson the concentration of a surfactant or that the activity hardly dependson the concentration of a surfactant was confirmed for different typesof lipoprotein lipase.

1. Preparation of Measurement Reagent

(Preparation of First Reagents)

1-1: Preparation of First Reagent (Reagent I)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent I ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mMN-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline 1.5 mM sodium salt(chromogen) Glycerol kinase 150 units/l Glycerol-3-phosphate oxidase3,000 units/l Sodium adenosine triphosphate 0.5 mM Magnesium chloridehexahydrate 1 mM Catalase 100,000 units/l BT-12 [polyoxyethylenesecondary alkyl ether in 0.1% (w/v) which the average mole number of theadded polyoxyethylene is 12] (Nikko Chemicals Co., Ltd)

1-2: Preparation of First Reagent (Reagent J)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent J ofpH 6.0 (20° C.). Regent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mMN-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline 1.5 mM sodium salt(chromogen) Glycerol kinase 150 units/l Glycerol-3-phosphate oxidase3,000 units/l Sodium adenosine triphosphate 0.5 mM Magnesium chloridehexahydrate 1 mM Catalase 100,000 units/l R-1020 [polyoxyethylenenonylphenyl formaldehyde 0.1% (w/v) condensate] (Nikko Chemicals Co.,Ltd)

(Preparation of Second Reagents)

1-3: Preparation of Second Reagent (Reagent K)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent K ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LP-BP (Asahi KaseiCorporation)] 100,000 units/l BT-12 [polyoxyethylene secondary alkylether in 0.1% (w/v) which the average mole number of the addedpolyoxyethylene is 12] (Nikko Chemicals Co., Ltd)

1-4: Preparation of Second Reagent (Reagent L)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent L ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LPL-314 (Toyobo Co., Ltd.)]100 units/l BT-12 [polyoxyethylene secondary alkyl ether in 0.1% (w/v)which the average mole number of the added polyoxyethylene is 12] (NikkoChemicals Co., Ltd)

1-5: Preparation of Second Reagent (Reagent M)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent M ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LPL (Asahi Kaseicorporation)] 100,000 units/l BT-12 [polyoxyethylene secondary alkylether in 0.1% (w/v) which the average mole number of the addedpolyoxyethylene is 12] (Nikko Chemicals Co., Ltd)

1-6: Preparation of Second Reagent (Reagent N)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent N ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LPL-311 (Toyobo Co., Ltd.)]100 units/l BT-12 [polyoxyethylene secondary alkyl ether in 0.1% (w/v)which the average mole number of the added polyoxyethylene is 12] (NikkoChemicals Co., Ltd)

1-7: Preparation of Second Reagent (Reagent 0)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent 0 ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LP-BP (Asahi KaseiCorporation)] 100,000 units/l R-1020 [polyoxyethylene nonylphenylformaldehyde 0.1% (w/v) condensate] (Nikko Chemicals Co., Ltd)

1-8: Preparation of Second Reagent (Reagent P)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent P ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LPL-314 (Toyobo Co., Ltd.)]100 units/l R-1020 [polyoxyethylene nonylphenyl formaldehyde 0.1% (w/v)condensate] (Nikko Chemicals Co., Ltd)

1-9: Preparation of Second Reagent (Reagent Q)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent Q ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LPL (Asahi KaseiCorporation)] 100,000 units/l R-1020 [polyoxyethylene nonylphenylformaldehyde 0.1% (w/v) condensate] (Nikko Chemicals Co., Ltd)

1-10: Preparation of Second Reagent (Reagent R)

The following reagent ingredients were dissolved in pure water to givethe respective indicated concentrations, thereby preparing Reagent R ofpH 6.0 (20° C.). Reagent ingredient Concentration2-Morpholinoethanesulfonic acid [MES] 50 mM 4-Aminoantipyrine 0.75 mMPeroxidase 600 units/l Lipoprotein lipase [LPL-311 (Toyobo Co., Ltd)]100 units/l R-1020 [polyoxyethylene nonylphenyl formaldehyde 0.1% (w/v)condensate] (Nikko Chemicals Co., Ltd)

2. Preparation of Purified Lipoprotein Fractions

Blood was collected in a tube containing an anticoagulant and subjectedto density-gradient centrifugation to obtain a very low densitylipoprotein fraction. This fraction was used as a test sample in themeasurement of triglycerides.

3. Confirmation of Lipoprotein Lipase's Character

The character of each lipoprotein lipase was confirmed by measuring,using the reagents prepared in section 1, triglycerides contained in thevery low density lipoprotein fraction prepared in section 2.

The measurement was made for the cases using the respective reagentcombinations shown in Table 6 in the same manner as in Example 1,section 4 “Measurement of triglycerides in lipoprotein fractions” usinga Hitachi automatic analyzer 7170S (Hitachi, Ltd.).

To confirm the character of each lipoprotein lipase, each of themeasured values [level of triglycerides contained in each test sample(very low density lipoprotein fraction)] “measured value” was divided byanother measured value as shown in Table 6.

The values are shown in Table 6. TABLE 6 Combinations of reagents FirstSecond reagent reagent (Lipoprotein lipase) Measurement 1 Reagent IReagent K (LP-BP) Measurement 2 Reagent I Reagent L (LPL-314)Measurement 3 Reagent I Reagent M (LPL) Measurement 4 Reagent I ReagentN (LPL-311) Measuratuion 5 Reagent J Reagent O (LP-BP) Measurement 6Reagent J Reagent P (LPL-314) Measurement 7 Reagent J Reagent Q (LPL)Measurement 8 Reagent J Reagent R (LPL-311) Values obtained by dividinga measured value by another measured value Surfactant: BT-12 Measurement1/measurement 0.15 3(LP-BP/LPL) Measurement 2/measurement 0.444(LP-314/LP-311) Surfactant: R-1020 Measurement 5/measurement 0.467(LP-BP/LPL) Measurement 6/measurement 0.29 8(LP-314/LP-311)

4. SUMMARY

4-1: When Surfactant is BT-12

1′. When dividing the measured value obtained using a measurementreagent whose second reagent contained LP-BP by the measured valueobtained using a measurement reagent whose second reagent contained LPL,the quotient was 0.15.

This indicates that the enzymatic activity of LPL was higher than thatof LP-BP when both measurement reagents contained the surfactant at thesame concentration.

This confirms that LP-BP is lipoprotein lipase of a type whose activitydepends on the concentration of the surfactant, while LPL is lipoproteinlipase of a type whose activity hardly depends on the concentration ofthe surfactant.

2′. When dividing the measured value obtained using a measurementreagent whose second reagent contained LPL-314 by the measured valueobtained using a measurement reagent whose second reagent containedLPL-311, the quotient was 0.44.

This indicates that the enzymatic activity of LPL-311 was higher thanthat of LPL-314 when both measurement reagents contained the surfactantat the same concentration.

This confirms that LPL-314 is lipoprotein lipase of a type whoseactivity depends on the concentration of the surfactant, while LPL-311is lipoprotein lipase of a type whose activity hardly depends on theconcentration of the surfactant.

4-2: When Surfactant is R-1020

1′. When dividing the measured value obtained using a measurementreagent whose second reagent contained LP-BP by the measured valueobtained using a measurement reagent whose second reagent contained LPL,the quotient was 0.46.

This indicates that the enzymatic activity of LPL was higher than thatof LP-BP when both measurement reagents contained the surfactant at thesame concentration.

This confirms that LP-BP is lipoprotein lipase of a type whose activitydepends on the concentration of the surfactant while LPL is lipoproteinlipase of a type whose activity hardly depends on the concentration ofthe surfactant.

2′. When dividing the measured value obtained using a measurementreagent whose second reagent contained LPL-314 by the measured valueobtained using a measurement reagent whose second reagent containedLPL-311, the quotient was 0.29.

This indicates that the enzymatic activity of LPL-311 was higher thanthat of LPL-314 when both measurement reagents contained the surfactantat the same concentration.

This confirms that LPL-314 is lipoprotein lipase of a type whoseactivity depends on the concentration of the surfactant, while LPL-311is lipoprotein lipase of a type whose activity hardly depends on theconcentration of the surfactant.

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The method and reagent for selective measurement of triglyceridescontained in very low density lipoprotein and intermediate densitylipoprotein or in very low density lipoprotein in a test sample inaccordance with this invention do not require labor and time consumingoperation, such as separating operation by ultracentrifuge, and isapplicable to automatic analyzers widely in use, thereby providing morereadily and accurately performable measurement.

1. A method for selective measurement of triglycerides contained in verylow density lipoprotein and intermediate density lipoprotein or in verylow density lipoprotein in a test sample, comprising the following twosteps: a first step which comprises 1′. exposing and reacting the testsample to and with lipoprotein lipase and some other enzymes, whichcatalyze a series of reactions leading to the generation of hydrogenperoxide or a reduced coenzyme from glycerol, in the presence of a firstselective reaction promoter, which is an ether or ester compound of apolyoxyalkylene capable of reacting lipoprotein lipase selectively withtriglycerides contained in low density lipoprotein and high densitylipoprotein, to generate hydrogen peroxide or a reduced coenzyme fromthe triglycerides contained in the low density lipoprotein and the highdensity lipoprotein in the test sample, 2′. reacting the hydrogenperoxide or reduced coenzyme generated by the reaction 1′ with an enzymewhich catalyzes a reaction leading to the conversion of hydrogenperoxide or a reduced coenzyme into another substance, and 3′.eliminating the triglycerides contained in the low density lipoproteinand the high density lipoprotein by the reactions of 1′ and 2′, and asecond step which comprises 1′. subsequently, after the first step,reacting the test sample with lipoprotein lipase and some other enzymes,which catalyze a series of reactions leading to the generation ofhydrogen peroxide or a reduced coenzyme from glycerol, in the presenceof a second selective reaction promoter, which is capable of reactinglipoprotein lipase selectively with triglycerides contained in very lowdensity lipoprotein, intermediate density lipoprotein, low densitylipoprotein and high density lipoprotein, to generate hydrogen peroxideor a reduced coenzyme from the triglycerides contained in the very lowdensity lipoprotein and the intermediate density lipoprotein or in thevery low density lipoprotein, and 2′. measuring the hydrogen peroxide orreduced coenzyme generated by the reaction 1′.
 2. The method accordingto claim 1, wherein the second selective reaction promoter is an etheror ester compound of a polyoxyalkylene.
 3. The method according to claim2, wherein m/n ratio is in the range of 1.1 to 1.2, where m is theaverage mole number of the added polyoxyalkylene in its ether or estercompound which is used as the first selective reaction promoter and n isthe average mole number of the added polyoxyalkylene in its ether orester compound which is used as the second selective reaction promoter.4. The method according to claim 3, wherein m is in the range of 7.7 to18 and n is in the range of 7 to
 15. 5. The method according to claim 3,wherein m is in the range of 11 to 12 and n is
 10. 6. The methodaccording to claim 1, wherein the ether or ester compound of apolyoxyalkylene which is used as the first selective reaction promoteris at least one selected from the group consisting of polyoxyalkylenestraight-chain alkyl ethers, polyoxyalkylene branched-chain alkylethers, polyoxyalkylene straight-chain alkylphenyl ethers,polyoxyalkylene branched-chain alkylphenyl ethers, polyoxyalkylenestraight-chain fatty acid esters, polyoxyalkylene branched-chain fattyacid esters, polyoxyalkylene straight-chain alkyl substituted benzoicacid esters and polyoxyalkylene branched-chain alkyl substituted benzoicacid esters.
 7. The method according to claim 1, wherein the secondselective reaction promoter is at least one ether or ester compound of apolyoxyalkylene selected from the group consisting of polyoxyalkylenestraight-chain alkyl ethers, polyoxyalkylene branched-chain alkylethers, polyoxyalkylene straight-chain alkylphenyl ethers,polyoxyalkylene branched-chain alkylphenyl ethers, polyoxyalkylenestraight-chain fatty acid esters, polyoxyalkylene branched-chain fattyacid esters, polyoxyalkylene straight-chain alkyl substituted benzoicacid esters and polyoxyalkylene branched-chain alkyl substituted benzoicacid esters.
 8. The method according to claim 1, wherein thepolyoxyalkylene is polyoxyethylene.
 9. The method according to claim 1,wherein the first selective reaction promoter is polyoxyethylenenonylphenyl ether in which the average mole number of addedpolyoxyethylene m is in the range of 11 to 12 and the second selectivereaction promoter is polyoxyethylene nonylphenyl ether in which theaverage mole number of added polyoxyethylene n is
 10. 10. The methodaccording to claim 1, wherein the first step and/or the second step iscarried out in the presence of a reaction assistant.
 11. The methodaccording to claim 10, wherein the reaction assistant is apolysaccharide or derivative thereof, a polyanion, a halogen ion, ametal ion, or lectin.
 12. The method according to claim 1, wherein theactivity of the lipoprotein lipase being present in the first stepdepends on the concentration of a surfactant, while that of thelipoprotein lipase being present in the second step hardly depends onthe concentration of a surfactant.
 13. A reagent for selectivemeasurement of triglycerides contained in very low density lipoproteinand intermediate density lipoprotein or in very low density lipoproteinin a test sample, comprising a first reagent that comprises: a firstselective reaction promoter, which is an ether or ester compound of apolyoxyalkylene capable of reacting lipoprotein lipase selectively withtriglycerides contained in low density lipoprotein and high densitylipoprotein; lipoprotein lipase; enzymes which catalyze a series ofreactions leading to the generation of hydrogen peroxide or a reducedcoenzyme from glycerol; and an enzyme which catalyzes a reaction leadingto the conversion of hydrogen peroxide or a reduced coenzyme intoanother substance, and a second reagent that comprises a secondselective reaction promoter, which is capable of reacting lipoproteinlipase selectively with triglycerides contained in very low densitylipoprotein, intermediate density lipoprotein, low density lipoproteinand high density lipoprotein.
 14. The reagent according to claim 13,wherein the first reagent and/or the second reagent further comprises asubstance which is involved in a reaction leading to the derivation ofsome signal from hydrogen peroxide or a reduced coenzyme.
 15. Thereagent according to claim 13, wherein the second selective reactionpromoter is an ether or ester compound of a polyoxyalkylene.
 16. Thereagent according to claim 15, wherein m/n ratio is in the range of 1.1to 1.2, where m is the average mole number of the added polyoxyalkylenein its ether or ester compound which is used as the first selectivereaction promoter and n is the average mole number of the addedpolyoxyalkylene in its ether or ester compound which is used as thesecond selective reaction promoter.
 17. The reagent according to claim16, wherein m is in the range of 7.7 to 18 and n is in the range of 7 to15.
 18. The reagent according to claim 16, wherein m is in the range of11 to 12 and n is
 10. 19. The reagent according to claim 13, wherein theether or ester compound of a polyoxyalkylene used as the first selectivereaction promoter is at least one selected from the group consisting ofpolyoxyalkylene straight-chain alkyl ethers, polyoxyalkylenebranched-chain alkyl ethers, polyoxyalkylene straight-chain alkylphenylethers, polyoxyalkylene branched-chain alkylphenyl ethers,polyoxyalkylene straight-chain fatty acid esters, polyoxyalkylenebranched-chain fatty acid esters, polyoxyalkylene straight-chain alkylsubstituted benzoic acid esters and polyoxyalkylene branched-chain alkylsubstituted benzoic acid esters.
 20. The reagent according to claim 13,wherein the second selective reaction promoter is at least one ether orester compound of a polyoxyalkylene selected from the group consistingof polyoxyalkylene straight-chain alkyl ethers, polyoxyalkylenebranched-chain alkyl ethers, polyoxyalkylene straight-chain alkylphenylethers, polyoxyalkylene branched-chain alkylphenyl ethers,polyoxyalkylene straight-chain fatty acid esters, polyoxyalkylenebranched-chain fatty acid esters, polyoxyalkylene straight-chain alkylsubstituted benzoic acid esters and polyoxyalkylene branched-chain alkylsubstituted benzoic acid esters.
 21. The reagent according to claim 13,wherein the polyoxyalkylene is polyoxyethylene.
 22. The reagentaccording to claim 13, wherein the first selective reaction promoter ispolyoxyethylene nonylphenyl ether in which the average mole number ofadded polyoxyethylene m is in the range of 11 to 12 and the secondselective reaction promoter is polyoxyethylene nonylphenyl ether inwhich the average mole number of added polyoxyethylene n is
 10. 23. Thereagent according to claim 13, wherein the first reagent and/or thesecond reagent further comprises a reaction assistant.
 24. The reagentaccording to claim 23, wherein the reaction assistant is apolysaccharide or derivative thereof, a polyanion, a halogen ion, ametal ion, or lectin.
 25. The reagent according to claim 13, wherein theactivity of the lipoprotein lipase contained in the first reagentdepends on the concentration of a surfactant, while that of thelipoprotein lipase contained in the second reagent hardly depends on theconcentration of a surfactant.